register interest

Professor Jane McKeating

Research Area: Microbiology
Scientific Themes: Immunology & Infectious Disease and Physiology, Cellular & Molecular Biology
Keywords: virus, hypoxia, infection and hepatitis

Our research focuses on understanding early infection events that define cellular and tissue tropism of clinically important viruses. Preventing viral induced disease begins with an understanding of the host factors that define susceptibility to infection.

Chronic viral infection of the liver is a global health problem, with over 500 million individuals infected with hepatitis B (HBV) or C (HCV) viruses that cause progressive liver disease that can develop into hepatocellular carcinoma. We recently discovered that the low oxygen environment naturally found in the liver enhances HBV and HCV replication and this is mediated by hypoxia inducible transcription factors (HIFs). Inhibiting HIFs or suppressing their expression limits virus infection, highlighting new therapeutic approaches.

Different anatomical sites experience varying oxygen tensions and we hypothesize that local changes in oxygen tension define active or latent states of viral replication. Immune cells encounter variable oxygen tensions as they migrate from the periphery into tissue and are a major target for HIV infection. Studies in our laboratory show that hypoxia limits HIV transcription and HIF-stabilizing drugs potentiate viral reactivation from latent sites of infection. Understanding the role of hypoxia induced signalling and metabolic effects on HIV transcription and latency will inform the development of new ‘curative strategies’.

HIFs coordinate extensive transcriptional responses that enable a cell to respond to diverse physiological or pathophysiological signals. Hepatitis B and C viruses activate HIFs and promote a ‘pseudohypoxic’ state that potentiates viral replication. Understanding how viruses influence host gene transcription is fundamental and profiling HIF-transcriptional targets in infected cells provides a unique opportunity to define the role of HIFs in viral-associated pathologies. 

Name Department Institution Country
Professor Sir Peter J Ratcliffe FRS Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Robert Thimme University Hospital Freiburg Germany
Professor Ulrike Protzer Institut für Virologie Technische Universität Munchen Germany
Professor Michael Wakelam (RDM) Babraham Institute United Kingdom
Professor Thomas Baumert Institute of Viral and Liver Disease University of Strasbourg France
Professor Greg Towers University College London United Kingdom
Professor Margeret Ashcroft Department of Medicine University of Cambridge United Kingdom
Professor Todd M Allen Ragon Institute of MGH Harvard University United States
Professor Josep llovet Hospital Clínic Barcelona. University of Barcelona Spain
Professor Charles M Rice Centre for HCV Research Rockefeller University United States
Ringelhan M, McKeating JA, Protzer U. 2017. Viral hepatitis and liver cancer. Philos Trans R Soc Lond B Biol Sci, 372 (1732), pp. 20160274-20160274. | Show Abstract | Read more

Hepatitis B and C viruses are a global health problem causing acute and chronic infections that can lead to liver cirrhosis and hepatocellular carcinoma (HCC). These infections are the leading cause for HCC worldwide and are associated with significant mortality, accounting for more than 1.3 million deaths per year. Owing to its high incidence and resistance to treatment, liver cancer is the second leading cause of cancer-related death worldwide, with HCC representing approximately 90% of all primary liver cancer cases. The majority of viral-associated HCC cases develop in subjects with liver cirrhosis; however, hepatitis B virus infection can promote HCC development without prior end-stage liver disease. Thus, understanding the role of hepatitis B and C viral infections in HCC development is essential for the future design of treatments and therapies for this cancer. In this review, we summarize the current knowledge on hepatitis B and C virus hepatocarcinogenesis and highlight direct and indirect risk factors.This article is part of the themed issue 'Human oncogenic viruses'.

Vettori A, Greenald D, Wilson GK, Peron M, Facchinello N, Markham E, Sinnakaruppan M, Matthews LC, McKeating JA, Argenton F, van Eeden FJM. 2017. Glucocorticoids promote Von Hippel Lindau degradation and Hif-1α stabilization. Proc Natl Acad Sci U S A, 114 (37), pp. 9948-9953. | Show Abstract | Read more

Glucocorticoid (GC) and hypoxic transcriptional responses play a central role in tissue homeostasis and regulate the cellular response to stress and inflammation, highlighting the potential for cross-talk between these two signaling pathways. We present results from an unbiased in vivo chemical screen in zebrafish that identifies GCs as activators of hypoxia-inducible factors (HIFs) in the liver. GCs activated consensus hypoxia response element (HRE) reporters in a glucocorticoid receptor (GR)-dependent manner. Importantly, GCs activated HIF transcriptional responses in a zebrafish mutant line harboring a point mutation in the GR DNA-binding domain, suggesting a nontranscriptional route for GR to activate HIF signaling. We noted that GCs increase the transcription of several key regulators of glucose metabolism that contain HREs, suggesting a role for GC/HIF cross-talk in regulating glucose homeostasis. Importantly, we show that GCs stabilize HIF protein in intact human liver tissue and isolated hepatocytes. We find that GCs limit the expression of Von Hippel Lindau protein (pVHL), a negative regulator of HIF, and that treatment with the c-src inhibitor PP2 rescued this effect, suggesting a role for GCs in promoting c-src-mediated proteosomal degradation of pVHL. Our data support a model for GCs to stabilize HIF through activation of c-src and subsequent destabilization of pVHL.

Farquhar MJ, Humphreys IS, Rudge SA, Wilson GK, Bhattacharya B, Ciaccia M, Hu K, Zhang Q, Mailly L, Reynolds GM et al. 2017. Autotaxin-lysophosphatidic acid receptor signalling regulates hepatitis C virus replication. J Hepatol, 66 (5), pp. 919-929. | Show Abstract | Read more

BACKGROUND & AIMS: Chronic hepatitis C is a global health problem with an estimated 170 million hepatitis C virus (HCV) infected individuals at risk of progressive liver disease and hepatocellular carcinoma (HCC). Autotaxin (ATX, gene name: ENPP2) is a phospholipase with diverse roles in the physiological and pathological processes including inflammation and oncogenesis. Clinical studies have reported increased ATX expression in chronic hepatitis C, however, the pathways regulating ATX and its role in the viral life cycle are not well understood. METHODS: In vitro hepatocyte and ex vivo liver culture systems along with chimeric humanized liver mice and HCC tissue enabled us to assess the interplay between ATX and the HCV life cycle. RESULTS: HCV infection increased hepatocellular ATX RNA and protein expression. HCV infection stabilizes hypoxia inducible factors (HIFs) and we investigated a role for these transcription factors to regulate ATX. In vitro studies show that low oxygen increases hepatocellular ATX expression and transcriptome analysis showed a positive correlation between ATX mRNA levels and hypoxia gene score in HCC tumour tissue associated with HCV and other aetiologies. Importantly, inhibiting ATX-lysophosphatidic acid (LPA) signalling reduced HCV replication, demonstrating a positive role for this phospholipase in the viral life cycle. LPA activates phosphoinositide-3-kinase that stabilizes HIF-1α and inhibiting the HIF signalling pathway abrogates the pro-viral activity of LPA. CONCLUSIONS: Our data support a model where HCV infection increases ATX expression which supports viral replication and HCC progression. LAY SUMMARY: Chronic hepatitis C is a global health problem with infected individuals at risk of developing liver disease that can progress to hepatocellular carcinoma. Autotaxin generates the biologically active lipid lysophosphatidic acid that has been reported to play a tumorigenic role in a wide number of cancers. In this study we show that hepatitis C virus infection increases autotaxin expression via hypoxia inducible transcription factor and provides an environment in the liver that promotes fibrosis and liver injury. Importantly, we show a new role for lysophosphatidic acid in positively regulating hepatitis C virus replication.

Gonçalves-Carneiro D, McKeating JA, Bailey D. 2017. The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis. J Virol, 91 (7), pp. e02255-16-e02255-16. | Show Abstract | Read more

The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen.IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between glycoproteins found on the surface of the virus and SLAMF1, the immune cell receptor. In this study, we have investigated the steps governing entry of measles virus into SLAMF1-positive cells and identified endocytic uptake of viral particles. This research will impact our understanding of morbillivirus-related immunosuppression as well as the application of measles virus as an oncolytic therapeutic.

Fletcher NF, Clark AR, Balfe P, McKeating JA. 2017. TNF superfamily members promote hepatitis C virus entry via an NF-κB and myosin light chain kinase dependent pathway. J Gen Virol, 98 (3), pp. 405-412. | Show Abstract | Read more

Preventing virally induced liver disease begins with an understanding of the host factors that define susceptibility to infection. Hepatitis C virus (HCV) is a global health issue, with an estimated 170 million infected individuals at risk of developing liver disease including fibrosis and hepatocellular carcinoma. The liver is the major reservoir supporting HCV replication and this hepatocellular tropism is defined by HCV engagement of cellular entry receptors. Hepatocytes are polarized in vivo and this barrier function limits HCV entry. We previously reported that activated macrophages promote HCV entry into polarized hepatocytes via a TNF-α-dependent process; however, the underlying mechanism was not defined. In this study, we show that several TNF superfamily members, including TNF-α, TNF-β, TWEAK and LIGHT, promote HCV entry via NF-κB-mediated activation of myosin light chain kinase (MLCK) and disruption of tight junctions. These observations support a model where HCV hijacks an inflammatory immune response to stimulate infection and uncovers a role for NF-κB-MLCK signalling in maintaining hepatocellular tight junctions.

Appelman MD, Chakraborty A, Protzer U, McKeating JA, van de Graaf SFJ. 2017. N-Glycosylation of the Na+-Taurocholate Cotransporting Polypeptide (NTCP) Determines Its Trafficking and Stability and Is Required for Hepatitis B Virus Infection. PLoS One, 12 (1), pp. e0170419. | Show Abstract | Read more

The sodium/bile acid cotransporter NTCP was recently identified as a receptor for hepatitis B virus (HBV). NTCP is glycosylated and the role of glycans in protein trafficking or viral receptor activity is not known. NTCP contains two N-linked glycosylation sites and asparagine amino acid residues N5 and N11 were mutated to a glutamine to generate NTCP with a single glycan (NTCP-N5Q or NTCP- N11Q) or no glycans (NTCP- N5,11Q). HepG2 cells expressing NTCP with a single glycan supported HBV infection at a comparable level to NTCP-WT. The physiological function of NTCP, the uptake of bile acids, was also not affected in cells expressing these single glycosylation variants, consistent with their trafficking to the plasma membrane. However, glycosylation-deficient NTCP (NTCP-N5,11Q) failed to support HBV infection, showed minimal cellular expression and was degraded in the lysosome. This affected the physiological bile acid transporter function of NTCP-N5,11Q in a similar fashion. In conclusion, N-glycosylation is required for efficient NTCP localization at the plasma membrane and subsequent HBV infection and these characteristics are preserved in NTCP carrying a single carbohydrate moiety.

Verrier ER, Colpitts CC, Bach C, Heydmann L, Zona L, Xiao F, Thumann C, Crouchet E, Gaudin R, Sureau C et al. 2016. Solute Carrier NTCP Regulates Innate Antiviral Immune Responses Targeting Hepatitis C Virus Infection of Hepatocytes. Cell Rep, 17 (5), pp. 1357-1368. | Show Abstract | Read more

Chronic hepatitis B, C, and D virus (HBV, HCV, and HDV) infections are the leading causes of liver disease and cancer worldwide. Recently, the solute carrier and sodium taurocholate co-transporter NTCP has been identified as a receptor for HBV and HDV. Here, we uncover NTCP as a host factor regulating HCV infection. Using gain- and loss-of-function studies, we show that NTCP mediates HCV infection of hepatocytes and is relevant for cell-to-cell transmission. NTCP regulates HCV infection by augmenting the bile-acid-mediated repression of interferon-stimulated genes (ISGs), including IFITM3. In conclusion, our results uncover NTCP as a mediator of innate antiviral immune responses in the liver, and they establish a role for NTCP in the infection process of multiple viruses via distinct mechanisms. Collectively, our findings suggest a role for solute carriers in the regulation of innate antiviral responses, and they have potential implications for virus-host interactions and antiviral therapies.

Tully DC, Hjerrild S, Leutscher PD, Renvillard SG, Ogilvie CB, Bean DJ, Videbech P, Allen TM, McKeating JA, Fletcher NF. 2016. Deep sequencing of hepatitis C virus reveals genetic compartmentalization in cerebrospinal fluid from cognitively impaired patients. Liver Int, 36 (10), pp. 1418-1424. | Show Abstract | Read more

BACKGROUND & AIMS: Hepatitis C virus (HCV) causes neuropsychiatric impairment and fatigue with recent studies suggesting HCV invasion of the central nervous system (CNS). Our previous finding that endothelial cells from the blood-brain barrier support HCV infection warrants further investigation to elucidate whether the CNS can serve as a reservoir for independent HCV evolution. METHODS: Cerebrospinal fluid (CSF) and plasma from six HCV-infected patients without liver disease or co-morbidities together with plasma from six healthy subjects were profiled for markers of immune activation and viral quasispecies measured by deep sequencing. Unsupervised data analyses were used to identify any associations between cytokine activation markers and clinical outcomes. RESULTS: Four of six HCV-infected patients showed significant evidence of cognitive dysfunction and fatigue. Deep sequencing revealed independent viral evolution within the CNS of two cognitively impaired patients. Principal component analysis of peripheral cytokines demonstrated that individuals without cognitive impairment clustered together while a distinct cytokine pattern emerged with patients exhibiting cognitive dysfunction and fatigue. CONCLUSIONS: Deep sequencing demonstrated unique viral variants in the CSF of two cognitively impaired patients consistent with CNS replication or sequestration. Meanwhile, compartmentalization was absent in infected patients with no neurocognitive impairment. Examination of cytokine profiles in HCV-infected patients with cognitive dysfunction revealed elevated peripheral cytokine levels resulting in a distinct cytokine profile that may be related to cognitive impairment or viral penetration into the CNS. Further studies to determine the significance of unique HCV variants within the CNS are warranted.

Hedegaard DL, Tully DC, Rowe IA, Reynolds GM, Bean DJ, Hu K, Davis C, Wilhelm A, Ogilvie CB, Power KA et al. 2017. High resolution sequencing of hepatitis C virus reveals limited intra-hepatic compartmentalization in end-stage liver disease. J Hepatol, 66 (1), pp. 28-38. | Show Abstract | Read more

BACKGROUND & AIMS: The high replication and mutation rate of hepatitis C virus (HCV) results in a heterogeneous population of viral sequences in vivo. HCV replicates in the liver and infected hepatocytes occur as foci surrounded by uninfected cells that may promote compartmentalization of viral variants. Given recent reports showing interferon stimulated gene (ISG) expression in chronic hepatitis C, we hypothesized that local interferon responses may limit HCV replication and evolution. METHODS: To investigate the spatial influence of liver architecture on viral replication we measured HCV RNA and ISG mRNA from each of the 8 Couinaud segments of the liver from 21 patients undergoing liver transplant. RESULTS: HCV RNA and ISG mRNA levels were comparable across all sites from an individual liver but showed up to 500-fold difference between patients. Importantly, there was no association between ISG and HCV RNA expression across all sites in the liver or plasma. Deep sequencing of HCV RNA isolated from the 8 hepatic sites from two subjects showed a similar distribution of viral quasispecies across the liver and uniform sequence diversity. Single genome amplification of HCV E1E2-envelope clones from 6 selected patients at 2 hepatic sites supported these data and showed no evidence for HCV compartmentalization. CONCLUSIONS: We found no differences between the hepatic and plasma viral quasispecies in all patients sampled. We conclude that in end-stage liver disease HCV RNA levels and the genetic pool of HCV envelope sequences are indistinguishable between distant sites in the liver and plasma, arguing against viral compartmentalization. LAY SUMMARY: HCV is an RNA virus that exists as a quasispecies of closely related genomes that are under continuous selection by host innate and adaptive immune responses and antiviral drug therapy. The primary site of HCV replication is the liver and yet our understanding of the spatial distribution of viral variants within the liver is limited. High resolution sequencing of HCV and monitoring of innate immune responses at multiple sites across the liver identified a uniform pattern of diversity and argues against viral compartmentalization.

Desombere I, Fafi-Kremer S, Van Houtte F, Pessaux P, Farhoudi A, Heydmann L, Verhoye L, Cole S, McKeating JA, Leroux-Roels G et al. 2016. Monoclonal anti-envelope antibody AP33 protects humanized mice against a patient-derived hepatitis C virus challenge. Hepatology, 63 (4), pp. 1120-1134. | Show Abstract | Read more

UNLABELLED: End-stage liver disease (ESLD) caused by hepatitis C virus (HCV) infection is a major indication for liver transplantation. However, immediately after transplantation, the liver graft of viremic patients universally becomes infected by circulating virus, resulting in accelerated liver disease progression. Currently available direct-acting antiviral therapies have reduced efficacy in patients with ESLD and prophylactic strategies to prevent HCV recurrence are still highly needed. In this study, we compared the ability of two broadly reactive monoclonal antibodies (mAbs), designated 3/11 and AP33, recognizing a distinct, but overlapping, epitope in the viral E2 glycoprotein to protect humanized mice from a patient-derived HCV challenge. Their neutralizing activity was assessed using the HCV pseudoparticles and cell-culture-derived HCV systems expressing multiple patient-derived envelopes and a human-liver chimeric mouse model. HCV RNA was readily detected in all control mice challenged with a patient-derived HCV genotype 1b isolate, whereas 3 of 4 AP33-treated mice were completely protected. In contrast, only one of four 3/11-treated mice remained HCV-RNA negative throughout the observation period, whereas the other 3 had a viral load that was indistinguishable from that in the control group. The increased in vivo efficacy of AP33 was in line with its higher affinity and neutralizing capacity observed in vitro. CONCLUSIONS: Although mAbs AP33 and 3/11 target the same region in E2, only mAb AP33 can efficiently protect from challenge with a heterologous HCV population in vivo. Given that mAb AP33 efficiently neutralizes viral variants that escaped the humoral immune response and reinfected the liver graft of transplant patients, it may be a valuable candidate to prevent HCV recurrence. In addition, our data are valuable for the design of a prophylactic vaccine.

Rowe IA, Tully DC, Armstrong MJ, Parker R, Guo K, Barton D, Morse GD, Venuto CS, Ogilvie CB, Hedegaard DL et al. 2016. Effect of scavenger receptor class B type I antagonist ITX5061 in patients with hepatitis C virus infection undergoing liver transplantation. Liver Transpl, 22 (3), pp. 287-297. | Show Abstract | Read more

Hepatitis C virus (HCV) entry inhibitors have been hypothesized to prevent infection of the liver after transplantation. ITX5061 is a scavenger receptor class B type I antagonist that blocks HCV entry and infection in vitro. We assessed the safety and efficacy of ITX5061 to limit HCV infection of the graft. The study included 23 HCV-infected patients undergoing liver transplantation. The first 13 "control" patients did not receive drug. The subsequent 10 patients received 150 mg of ITX5061 immediately before and after transplant and daily for 1 week thereafter. ITX5061 pharmacokinetics and plasma HCV RNA were quantified. Viral genetic diversity was measured by ultradeep pyrosequencing (UDPS). ITX5061 was well tolerated with measurable plasma concentrations during therapy. Although the median HCV RNA reduction was greater in ITX-treated patients at all time points in the first week after transplantation, there was no difference in the overall change in the area over the HCV RNA curve in the 7-day treatment period. However, in genotype (GT) 1-infected patients, treatment was associated with a sustained reduction in HCV RNA levels compared to the control group (area over the HCV RNA curve analysis, P = 0.004). UDPS revealed a complex and evolving pattern of HCV variants infecting the graft during the first week. ITX5061 significantly limited viral evolution where the median divergence between day 0 and day 7 was 3.5% in the control group compared to 0.1% in the treated group. In conclusion, ITX5061 reduces plasma HCV RNA after transplant notably in GT 1-infected patients and slows viral evolution. Following liver transplantation, the likely contribution of extrahepatic reservoirs of HCV necessitates combining entry inhibitors such as ITX5061 with inhibitors of replication in future studies.

Zhang Y-H, Zhao Y, Rajapaksa US, Lawrence TM, Peng Y-C, Liu J, Xu K, Hu K, Qin L, Liu N et al. 2016. A Comprehensive Analysis of the Impact of HIV on HCV Immune Responses and Its Association with Liver Disease Progression in a Unique Plasma Donor Cohort. PLoS One, 11 (7), pp. e0158037. | Show Abstract | Read more

OBJECTIVE: Human Immunodeficiency Virus (HIV) and Hepatitis C virus (HCV) co-infection is recognized as a major cause of morbidity and mortality among HIV-1 infected patients. Our understanding of the impact of HIV infection on HCV specific immune responses and liver disease outcome is limited by the heterogeneous study populations with genetically diverse infecting viruses, varying duration of infection and anti-viral treatment. METHODS: Viral-specific immune responses in a cohort of 151 HCV mono- and HIV co-infected former plasma donors infected with a narrow source of virus were studied. HCV and HIV specific T cell responses were correlated with clinical data. RESULTS: HIV-1 accelerated liver disease progression and decreased HCV specific T cell immunity. The magnitude of HCV specific T cell responses inversely correlated with lower HCV RNA load and reduced liver injury as assessed by non-invasive markers of liver fibrosis. HIV co-infection reduced the frequency of HCV specific CD4+ T cells with no detectable effect on CD8+ T cells or neutralizing antibody levels. CONCLUSION: Our study highlights the impact of HIV co-infection on HCV specific CD4+ T cell responses in a unique cohort of patients for both HCV and HIV and suggests a crucial role for these cells in controlling chronic HCV replication and liver disease progression.

Meredith LW, Hu K, Cheng X, Howard CR, Baumert TF, Balfe P, van de Graaf KF, Protzer U, McKeating JA. 2016. Lentiviral hepatitis B pseudotype entry requires sodium taurocholate co-transporting polypeptide and additional hepatocyte-specific factors. J Gen Virol, 97 (1), pp. 121-127. | Show Abstract | Read more

Hepatitis B virus (HBV) is one of the world's major unconquered infections, resulting in progressive liver disease, and current treatments rarely cure infection. A limitation to discovering new therapies is our limited knowledge of HBV entry and dissemination pathways that hinders the development of in vitro culture systems. To address this gap in our understanding we optimized the genesis of infectious lentiviral pseudoparticles (HBVpps). The recent discovery that the bile salt transporter sodium taurocholate co-transporting polypeptide (NTCP) acts as a receptor for HBV enabled us to assess the receptor dependency of HBVpp infection. HBVpps preferentially infect hepatoma cells expressing NTCP, whereas other non-liver cells engineered to express NTCP do not support infection, suggesting that additional hepatocyte-specific factors are required for HBVpp internalization. These results highlight the value of the HBVpp system to dissect the pathways of HBV entry and dissemination.

Mathiesen CK, Prentoe J, Meredith LW, Jensen TB, Krarup H, McKeating JA, Gottwein JM, Bukh J. 2015. Adaptive Mutations Enhance Assembly and Cell-to-Cell Transmission of a High-Titer Hepatitis C Virus Genotype 5a Core-NS2 JFH1-Based Recombinant. J Virol, 89 (15), pp. 7758-7775. | Show Abstract | Read more

UNLABELLED: Recombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here, we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1C3405G,A3696G (termed SA13/JFH1orig), of the poorly characterized genotype 5a, to Huh7.5 cells, yielding a virus with greatly improved spread kinetics and an infectivity titer of 6.7 log10 focus-forming units (FFU)/ml. We identified several putative adaptive amino acid changes. In head-to-head infections at fixed multiplicities of infection, one SA13/JFH1orig mutant termed SA13/JFH1Core-NS5B, containing 13 amino acid changes (R114W and V187A [Core]; V235L [E1]; T385P [E2]; L782V [p7]; Y900C [NS2]; N2034D, E2238G, V2252A, L2266P, and I2340T [NS5A]; A2500S and V2841A [NS5B]), displayed fitness comparable to that of the polyclonal high-titer adapted virus. Single-cycle virus production assays in CD81-deficient Huh7-derived cells demonstrated that these changes did not affect replication but increased HCV assembly and specific infectivity as early as 24 h posttransfection. Infectious coculture assays in Huh7.5 cells showed a significant increase in cell-to-cell transmission for SA13/JFH1Core-NS5B viruses as well as viruses with only p7 and nonstructural protein mutations. Interestingly, the E2 hypervariable region 1 (HVR1) mutation T385P caused (i) increased sensitivity to neutralizing patient IgG and human monoclonal antibodies AR3A and AR4A and (ii) increased accessibility of the CD81 binding site without affecting the usage of CD81 and SR-BI. We finally demonstrated that SA13/JFH1orig and SA13/JFH1Core-NS5B, with and without the E2 mutation T385P, displayed similar biophysical properties following iodixanol gradient ultracentrifugation. This study has implications for investigations requiring high virus concentrations, such as studies of HCV particle composition and development of whole-virus vaccine antigens. IMPORTANCE: Hepatitis C virus (HCV) is a major global health care burden, affecting more than 150 million people worldwide. These individuals are at high risk of developing severe end-stage liver diseases. No vaccine exists. While it is possible to produce HCV particles resembling isolates of all HCV genotypes in human hepatoma cells (HCVcc), production efficacy varies. Thus, for several important studies, including vaccine development, in vitro systems enabling high-titer production of diverse HCV strains would be advantageous. Our study offers important functional data on how cell culture-adaptive mutations identified in genotype 5a JFH1-based HCVcc permit high-titer culture by affecting HCV genesis through increasing virus assembly and HCV fitness by enhancing the virus specific infectivity and cell-to-cell transmission ability, without influencing the biophysical particle properties. High-titer HCVcc like the one described in this study may be pivotal in future vaccine-related studies where large quantities of infectious HCV particles are necessary.

Fletcher NF, Humphreys E, Jennings E, Osburn W, Lissauer S, Wilson GK, van IJzendoorn SCD, Baumert TF, Balfe P, Afford S, McKeating JA. 2015. Hepatitis C virus infection of cholangiocarcinoma cell lines. J Gen Virol, 96 (Pt 6), pp. 1380-1388. | Show Abstract | Read more

Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV replication and contribute to the hepatic reservoir. We screened cholangiocytes along with a panel of cholangiocarcinoma-derived cell lines for their ability to support HCV entry and replication. While primary cholangiocytes were refractory to infection and lacked expression of several entry factors, two cholangiocarcinoma lines, CC-LP-1 and Sk-ChA-1, supported efficient HCV entry; furthermore, Sk-ChA-1 cells supported full virus replication. In vivo cholangiocarcinomas expressed all of the essential HCV entry factors; however, cholangiocytes adjacent to the tumour and in normal tissue showed a similar pattern of receptor expression to ex vivo isolated cholangiocytes, lacking SR-BI expression, explaining their inability to support infection. This study provides the first report that HCV can infect cholangiocarcinoma cells and suggests that these heterogeneous tumours may provide a reservoir for HCV replication in vivo.

Wilson GK, Farquhar MJ, Meredith L, Dhawan A, Mitry R, Balfe P, McKeating JA. 2015. Permissivity of primary hepatocytes and hepatoma cell lines to support hepatitis C virus infection. J Gen Virol, 96 (Pt 6), pp. 1369-1373. | Show Abstract | Read more

The major cell type supporting hepatitis C virus (HCV) infection is the hepatocyte; however, most reports studying viral entry and replication utilize transformed hepatoma cell lines. We demonstrate that HCV pseudoparticles (HCVpp) infect primary hepatocytes with comparable rates to hepatoma cells, demonstrating the limited variability in donor hepatocytes to support HCV receptor-glycoprotein-dependent entry. In contrast, we observed a 2-log range in viral replication between the same donor hepatocytes. We noted that cell proliferation augments pseudoparticle reporter activity and arresting hepatoma cells yields comparable levels of infection to hepatocytes. This study demonstrates comparable rates of HCVpp entry into primary hepatocytes and hepatoma cells, validating the use of transformed cells as a model system to study HCV entry.

Mailly L, Xiao F, Lupberger J, Wilson GK, Aubert P, Duong FHT, Calabrese D, Leboeuf C, Fofana I, Thumann C et al. 2015. Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody. Nat Biotechnol, 33 (5), pp. 549-554. | Show Abstract | Read more

Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer. Cell entry of HCV and other pathogens is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model, we show that a monoclonal antibody specific for the TJ protein claudin-1 (ref. 7) eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection by means of host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy.

Meola A, Tarr AW, England P, Meredith LW, McClure CP, Foung SKH, McKeating JA, Ball JK, Rey FA, Krey T. 2015. Structural flexibility of a conserved antigenic region in hepatitis C virus glycoprotein E2 recognized by broadly neutralizing antibodies. J Virol, 89 (4), pp. 2170-2181. | Show Abstract | Read more

UNLABELLED: Neutralizing antibodies (NAbs) targeting glycoprotein E2 are important for the control of hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412 to 423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a β-hairpin in complex with three independent NAbs. Our structure of the same peptide in complex with NAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412 to 423 are essential for virus entry but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab fragment, this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and β-hairpin conformations, respectively) display similar neutralizing activities despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as an immune evasion strategy, contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and nonenveloped viruses. IMPORTANCE: Approximately 180 million people worldwide are infected with hepatitis C virus (HCV), and neutralizing antibodies play an important role in controlling the replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412 to 423), which is disordered in the recently reported crystal structure of an E2 core fragment, can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activities in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than those targeting other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic region contributes to the evasion of the humoral host immune response, facilitating chronicity and the viral spread of HCV within an infected individual.

Wilson GK, Tennant DA, McKeating JA. 2014. Hypoxia inducible factors in liver disease and hepatocellular carcinoma: current understanding and future directions. J Hepatol, 61 (6), pp. 1397-1406. | Show Abstract | Read more

Hypoxia inducible transcription factors (HIFs) activate diverse pathways that regulate cellular metabolism, angiogenesis, proliferation, and migration, enabling a cell to respond to a low oxygen or hypoxic environment. HIFs are regulated by oxygen-dependent and independent signals including: mitochondrial dysfunction, reactive oxygen species, endoplasmic reticular stress, and viral infection. HIFs have been reported to play a role in the pathogenesis of liver disease of diverse aetiologies. This review explores the impact of HIFs on hepatocellular biology and inflammatory responses, highlighting the therapeutic potential of targeting HIFs for an array of liver pathologies.

Meredith LW, Farquhar MJ, Tarr AW, McKeating JA. 2014. Type I interferon rapidly restricts infectious hepatitis C virus particle genesis. Hepatology, 60 (6), pp. 1891-1901. | Show Abstract | Read more

UNLABELLED: Interferon-alpha (IFNα) has been used to treat chronic hepatitis C virus (HCV) infection for over 20 years with varying efficacy, depending on the infecting viral genotype. The mechanism of action of IFNα is not fully understood, but is thought to target multiple stages of the HCV lifecycle, inhibiting viral transcription and translation leading to a degradation of viral RNA and protein expression in the infected cell. IFNα induces the expression of an array of interferon-stimulated genes within minutes of receptor engagement; however, the impact of these early responses on the viral lifecycle are unknown. We demonstrate that IFNα inhibits the genesis of infectious extracellular HCV particles within 2 hours of treating infected cells, with minimal effect on the intracellular viral burden. Importantly, this short duration of IFNα treatment of infected cells significantly reduced cell-free and cell-to-cell dissemination. The secreted viral particles showed no apparent change in protein content or density, demonstrating that IFNα inhibits particle infectivity but not secretion rates. To investigate whether particles released from IFNα-treated cells have a reduced capacity to establish infection we used HCV lentiviral pseudotypes (HCVpp) and demonstrated a defect in cell entry. Using a panel of monoclonal antibodies targeting the E2 glycoprotein, we demonstrate that IFNα alters glycoprotein conformation and receptor utilization. CONCLUSION: These observations show a previously unreported and rapid effect of IFNα on HCV particle infectivity that inhibits de novo infection events. Evasion of this response may be a contributing factor in whether a patient achieves early or rapid virological response, a key indicator of progression to sustained virological response or clearance of viral infection.

Ball JK, Tarr AW, McKeating JA. 2014. The past, present and future of neutralizing antibodies for hepatitis C virus. Antiviral Res, 105 (1), pp. 100-111. | Show Abstract | Read more

Hepatitis C virus (HCV) is a major cause of liver disease and hepatocellular carcinoma worldwide. HCV establishes a chronic infection in the majority of cases. However, some individuals clear the virus, demonstrating a protective role for the host immune response. Although new all-oral drug combinations may soon replace traditional ribavirin-interferon therapy, the emerging drug cocktails will be expensive and associated with side-effects and resistance, making a global vaccine an urgent priority. T cells are widely accepted to play an essential role in clearing acute HCV infection, whereas the role antibodies play in resolution and disease pathogenesis is less well understood. Recent studies have provided an insight into viral neutralizing determinants and the protective role of antibodies during infection. This review provides a historical perspective of the role neutralizing antibodies play in HCV infection and discusses the therapeutic benefits of antibody-based therapies. This article forms part of a symposium in Antiviral Research on "Hepatitis C: next steps toward global eradication."

Chmielewska AM, Naddeo M, Capone S, Ammendola V, Hu K, Meredith L, Verhoye L, Rychlowska M, Rappuoli R, Ulmer JB et al. 2014. Combined adenovirus vector and hepatitis C virus envelope protein prime-boost regimen elicits T cell and neutralizing antibody immune responses. J Virol, 88 (10), pp. 5502-5510. | Show Abstract | Read more

UNLABELLED: Despite the recent progress in the development of new antiviral agents, hepatitis C virus (HCV) infection remains a major global health problem, and there is a need for a preventive vaccine. We previously reported that adenoviral vectors expressing HCV nonstructural proteins elicit protective T cell responses in chimpanzees and were immunogenic in healthy volunteers. Furthermore, recombinant HCV E1E2 protein formulated with adjuvant MF59 induced protective antibody responses in chimpanzees and was immunogenic in humans. To develop an HCV vaccine capable of inducing both T cell and antibody responses, we constructed adenoviral vectors expressing full-length and truncated E1E2 envelope glycoproteins from HCV genotype 1b. Heterologous prime-boost immunization regimens with adenovirus and recombinant E1E2 glycoprotein (genotype 1a) plus MF59 were evaluated in mice and guinea pigs. Adenovirus prime and protein boost induced broad HCV-specific CD8+ and CD4+ T cell responses and functional Th1-type IgG responses. Immune sera neutralized luciferase reporter pseudoparticles expressing HCV envelope glycoproteins (HCVpp) and a diverse panel of recombinant cell culture-derived HCV (HCVcc) strains and limited cell-to-cell HCV transmission. This study demonstrated that combining adenovirus vector with protein antigen can induce strong antibody and T cell responses that surpass immune responses achieved by either vaccine alone. IMPORTANCE: HCV infection is a major health problem. Despite the availability of new directly acting antiviral agents for treating chronic infection, an affordable preventive vaccine provides the best long-term goal for controlling the global epidemic. This report describes a new anti-HCV vaccine targeting the envelope viral proteins based on adenovirus vector and protein in adjuvant. Rodents primed with the adenovirus vaccine and boosted with the adjuvanted protein developed cross-neutralizing antibodies and potent T cell responses that surpassed immune responses achieved with either vaccine component alone. If combined with the adenovirus vaccine targeting the HCV NS antigens now under clinical testing, this new vaccine might lead to a stronger and broader immune response and to a more effective vaccine to prevent HCV infection. Importantly, the described approach represents a valuable strategy for other infectious diseases in which both T and B cell responses are essential for protection.

Colley HE, Hearnden V, Avila-Olias M, Cecchin D, Canton I, Madsen J, MacNeil S, Warren N, Hu K, McKeating JA et al. 2014. Polymersome-mediated delivery of combination anticancer therapy to head and neck cancer cells: 2D and 3D in vitro evaluation. Mol Pharm, 11 (4), pp. 1176-1188. | Show Abstract | Read more

Polymersomes have the potential to encapsulate and deliver chemotherapeutic drugs into tumor cells, reducing off-target toxicity that often compromises anticancer treatment. Here, we assess the ability of the pH-sensitive poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC)- poly 2-(diisopropylamino)ethyl methacrylate (PDPA) polymersomes to encapsulate chemotherapeutic agents for effective combinational anticancer therapy. Polymersome uptake and ability to deliver encapsulated drugs into healthy normal oral cells and oral head and neck squamous cell carcinoma (HNSCC) cells was measured in two and three-dimensional culture systems. PMPC-PDPA polymersomes were more rapidly internalized by HNSCC cells compared to normal oral cells. Polymersome cellular uptake was found to be mediated by class B scavenger receptors. We also observed that these receptors are more highly expressed by cancer cells compared to normal oral cells, enabling polymersome-mediated targeting. Doxorubicin and paclitaxel were encapsulated into pH-sensitive PMPC-PDPA polymersomes with high efficiencies either in isolation or as a dual-load for both singular and combinational delivery. In monolayer culture, only a short exposure to drug-loaded polymersomes was required to elicit a strong cytotoxic effect. When delivered to three-dimensional tumor models, PMPC-PDPA polymersomes were able to penetrate deep into the center of the spheroid resulting in extensive cell damage when loaded with both singular and dual-loaded chemotherapeutics. PMPC-PDPA polymersomes offer a novel system for the effective delivery of chemotherapeutics for the treatment of HNSCC. Moreover, the preferential internalization of PMPC polymersomes by exploiting elevated scavenger receptor expression on cancer cells opens up the opportunity to target polymersomes to tumors.

Fletcher NF, Sutaria R, Jo J, Barnes A, Blahova M, Meredith LW, Cosset F-L, Curbishley SM, Adams DH, Bertoletti A, McKeating JA. 2014. Activated macrophages promote hepatitis C virus entry in a tumor necrosis factor-dependent manner. Hepatology, 59 (4), pp. 1320-1330. | Show Abstract | Read more

UNLABELLED: Macrophages are critical components of the innate immune response in the liver. Chronic hepatitis C is associated with immune infiltration and the infected liver shows a significant increase in total macrophage numbers; however, their role in the viral life cycle is poorly understood. Activation of blood-derived and intrahepatic macrophages with a panel of Toll-like receptor agonists induce soluble mediators that promote hepatitis C virus (HCV) entry into polarized hepatoma cells. We identified tumor necrosis factor α (TNF-α) as the major cytokine involved in this process. Importantly, this effect was not limited to HCV; TNF-α increased the permissivity of hepatoma cells to infection by Lassa, measles and vesicular stomatitis pseudoviruses. TNF-α induced a relocalization of tight junction protein occludin and increased the lateral diffusion speed of HCV receptor tetraspanin CD81 in polarized HepG2 cells, providing a mechanism for their increased permissivity to support HCV entry. High concentrations of HCV particles could stimulate macrophages to express TNF-α, providing a direct mechanism for the virus to promote infection. CONCLUSION: This study shows a new role for TNF-α to increase virus entry and highlights the potential for HCV to exploit existing innate immune responses in the liver to promote de novo infection events.

Baumert TF, Meredith L, Ni Y, Felmlee DJ, McKeating JA, Urban S. 2014. Entry of hepatitis B and C viruses - recent progress and future impact. Curr Opin Virol, 4 pp. 58-65. | Show Abstract | Read more

Chronic hepatitis B and C virus infections are major causes of liver disease and hepatocellular carcinoma worldwide. Although both viruses infect hepatocytes, the molecular virology and cellular biology of their respective replication cycles differ. Viral entry is the first step of the life cycle and recent developments in functional genomic and proteomic methodologies have increased our understanding of the entry pathways for these two important human pathogens. In this review we provide a comparative analysis of the internalization routes for these viruses and highlight differences and how they impact the viral life cycle, immune responses and development of antivirals.

Marsh M, McKeating JA. 2014. Editorial overview - virus entry: towards reality - refining models of virus entry. Curr Opin Virol, 4 pp. v-vi. | Read more

Rowe IA, Galsinh SK, Wilson GK, Parker R, Durant S, Lazar C, Branza-Nichita N, Bicknell R, Adams DH, Balfe P, McKeating JA. 2014. Paracrine signals from liver sinusoidal endothelium regulate hepatitis C virus replication. Hepatology, 59 (2), pp. 375-384. | Show Abstract | Read more

UNLABELLED: Hepatitis C virus (HCV) is a major cause of global morbidity, causing chronic liver injury that can progress to cirrhosis and hepatocellular carcinoma. The liver is a large and complex organ containing multiple cell types, including hepatocytes, sinusoidal endothelial cells (LSEC), Kupffer cells, and biliary epithelial cells. Hepatocytes are the major reservoir supporting HCV replication; however, the role of nonparenchymal cells in the viral lifecycle remains largely unexplored. LSEC secrete factors that promote HCV infection and transcript analysis identified bone morphogenetic protein 4 (BMP4) as a candidate endothelial-expressed proviral molecule. Recombinant BMP4 increased HCV replication and neutralization of BMP4 abrogated the proviral activity of LSEC-conditioned media. Importantly, BMP4 expression was negatively regulated by vascular endothelial growth factor A (VEGF-A) by way of a VEGF receptor-2 (VEGFR-2) primed activation of p38 MAPK. Consistent with our in vitro observations, we demonstrate that in normal liver VEGFR-2 is activated and BMP4 expression is suppressed. In contrast, in chronic liver disease including HCV infection where there is marked endothelial cell proliferation, we observed reduced endothelial cell VEGFR-2 activation and a concomitant increase in BMP4 expression. CONCLUSION: These studies identify a role for LSEC and BMP4 in HCV infection and highlight BMP4 as a new therapeutic target for treating individuals with liver disease.

Hwang K-K, Trama AM, Kozink DM, Chen X, Wiehe K, Cooper AJ, Xia S-M, Wang M, Marshall DJ, Whitesides J et al. 2014. IGHV1-69 B cell chronic lymphocytic leukemia antibodies cross-react with HIV-1 and hepatitis C virus antigens as well as intestinal commensal bacteria. PLoS One, 9 (3), pp. e90725. | Show Abstract | Read more

B-cell chronic lymphocytic leukemia (B-CLL) patients expressing unmutated immunoglobulin heavy variable regions (IGHVs) use the IGHV1-69 B cell receptor (BCR) in 25% of cases. Since HIV-1 envelope gp41 antibodies also frequently use IGHV1-69 gene segments, we hypothesized that IGHV1-69 B-CLL precursors may contribute to the gp41 B cell response during HIV-1 infection. To test this hypothesis, we rescued 5 IGHV1-69 unmutated antibodies as heterohybridoma IgM paraproteins and as recombinant IgG1 antibodies from B-CLL patients, determined their antigenic specificities and analyzed BCR sequences. IGHV1-69 B-CLL antibodies were enriched for reactivity with HIV-1 envelope gp41, influenza, hepatitis C virus E2 protein and intestinal commensal bacteria. These IGHV1-69 B-CLL antibodies preferentially used IGHD3 and IGHJ6 gene segments and had long heavy chain complementary determining region 3s (HCDR3s) (≥21 aa). IGHV1-69 B-CLL BCRs exhibited a phenylalanine at position 54 (F54) of the HCDR2 as do rare HIV-1 gp41 and influenza hemagglutinin stem neutralizing antibodies, while IGHV1-69 gp41 antibodies induced by HIV-1 infection predominantly used leucine (L54) allelic variants. These results demonstrate that the B-CLL cell population is an expansion of members of the innate polyreactive B cell repertoire with reactivity to a number of infectious agent antigens including intestinal commensal bacteria. The B-CLL IGHV1-69 B cell usage of F54 allelic variants strongly suggests that IGHV1-69 B-CLL gp41 antibodies derive from a restricted B cell pool that also produces rare HIV-1 gp41 and influenza hemagglutinin stem antibodies.

Brimacombe CL, Wilson GK, Hübscher SG, McKeating JA, Farquhar MJ. 2014. A role for CD81 and hepatitis C virus in hepatoma mobility. Viruses, 6 (3), pp. 1454-1472. | Show Abstract | Read more

Tetraspanins are a family of small proteins that interact with themselves, host transmembrane and cytosolic proteins to form tetraspanin enriched microdomains (TEMs) that regulate important cellular functions. Several tetraspanin family members are linked to tumorigenesis. Hepatocellular carcinoma (HCC) is an increasing global health burden, in part due to the increasing prevalence of hepatitis C virus (HCV) associated HCC. The tetraspanin CD81 is an essential receptor for HCV, however, its role in hepatoma biology is uncertain. We demonstrate that antibody engagement of CD81 promotes hepatoma spread, which is limited by HCV infection, in an actin-dependent manner and identify an essential role for the C-terminal interaction with Ezrin-Radixin-Moesin (ERM) proteins in this process. We show enhanced hepatoma migration and invasion following expression of CD81 and a reduction in invasive potential upon CD81 silencing. In addition, we reveal poorly differentiated HCC express significantly higher levels of CD81 compared to adjacent non-tumor tissue. In summary, these data support a role for CD81 in regulating hepatoma mobility and propose CD81 as a tumour promoter.

Meredith LW, Zitzmann N, McKeating JA. 2013. Differential effect of p7 inhibitors on hepatitis C virus cell-to-cell transmission. Antiviral Res, 100 (3), pp. 636-639. | Show Abstract | Read more

Inhibitors targeting the hepatitis C virus (HCV) encoded viroporin, p7 prevent virus release in vitro. HCV can transmit by cell-free particle infection of new target cells and via cell-to-cell dependent contact with limited exposure to the extracellular environment. The role of assembly inhibitors in preventing HCV transmission via these pathways has not been studied. We compared the efficacy of three published p7 inhibitors to inhibit cell-free and cell-to-cell transmission of two chimeric HCV strains encoding genotype 2 (GT2) or 5 (GT5) p7 using a recently developed single cycle co-culture assay. The inhibitors reduced the infectivity of extracellular GT2 and GT5 virus by 80-90% and GT2 virus cell-to-cell transmission by 50%. However, all of the p7 inhibitors had minimal effect on GT5 cell contact dependent transmission. Screening a wider panel of diverse viral genotypes demonstrated that p7 viroporin inhibitors were significantly more effective at blocking cell-free virus than cell-to-cell transmission. These results suggest an altered assembly or trafficking of cell-to-cell transmitted compared to secreted virus. These observations have important implications for the validation, therapeutic design and testing of HCV assembly inhibitors.

McKeating JA, Howard CR. 2013. Viral hepatitis and hepatocellular carcinoma Microbiology Today, 40 (1), pp. 30-33.

Tarr AW, Lafaye P, Meredith L, Damier-Piolle L, Urbanowicz RA, Meola A, Jestin J-L, Brown RJP, McKeating JA, Rey FA et al. 2013. An alpaca nanobody inhibits hepatitis C virus entry and cell-to-cell transmission. Hepatology, 58 (3), pp. 932-939. | Show Abstract | Read more

UNLABELLED: Severe liver disease caused by chronic hepatitis C virus is the major indication for liver transplantation. Despite recent advances in antiviral therapy, drug toxicity and unwanted side effects render effective treatment in liver-transplanted patients a challenging task. Virus-specific therapeutic antibodies are generally safe and well-tolerated, but their potential in preventing and treating hepatitis C virus (HCV) infection has not yet been realized due to a variety of issues, not least high production costs and virus variability. Heavy-chain antibodies or nanobodies, produced by camelids, represent an exciting antiviral approach; they can target novel highly conserved epitopes that are inaccessible to normal antibodies, and they are also easy to manipulate and produce. We isolated four distinct nanobodies from a phage-display library generated from an alpaca immunized with HCV E2 glycoprotein. One of them, nanobody D03, recognized a novel epitope overlapping with the epitopes of several broadly neutralizing human monoclonal antibodies. Its crystal structure revealed a long complementarity determining region (CD3) folding over part of the framework that, in conventional antibodies, forms the interface between heavy and light chain. D03 neutralized a panel of retroviral particles pseudotyped with HCV glycoproteins from six genotypes and authentic cell culture-derived particles by interfering with the E2-CD81 interaction. In contrast to some of the most broadly neutralizing human anti-E2 monoclonal antibodies, D03 efficiently inhibited HCV cell-to-cell transmission. CONCLUSION: This is the first description of a potent and broadly neutralizing HCV-specific nanobody representing a significant advance that will lead to future development of novel entry inhibitors for the treatment and prevention of HCV infection and help our understanding of HCV cell-to-cell transmission.

Fofana I, Zona L, Thumann C, Heydmann L, Durand SC, Lupberger J, Blum HE, Pessaux P, Gondeau C, Reynolds GM et al. 2013. Functional analysis of claudin-6 and claudin-9 as entry factors for hepatitis C virus infection of human hepatocytes by using monoclonal antibodies. J Virol, 87 (18), pp. 10405-10410. | Show Abstract | Read more

The relevance of claudin-6 and claudin-9 in hepatitis C virus (HCV) entry remains elusive. We produced claudin-6- or claudin-9-specific monoclonal antibodies that inhibit HCV entry into nonhepatic cells expressing exogenous claudin-6 or claudin-9. These antibodies had no effect on HCV infection of hepatoma cells or primary hepatocytes. Thus, although claudin-6 and claudin-9 can serve as entry factors in cell lines, HCV infection into human hepatocytes is not dependent on claudin-6 and claudin-9.

Xiao F, McKeating JA, Baumert TF. 2013. A bile acid transporter as a candidate receptor for hepatitis B and D virus entry. J Hepatol, 58 (6), pp. 1246-1248. | Read more

Meredith LW, Harris HJ, Wilson GK, Fletcher NF, Balfe P, McKeating JA. 2013. Early infection events highlight the limited transmissibility of hepatitis C virus in vitro. J Hepatol, 58 (6), pp. 1074-1080. | Show Abstract | Read more

BACKGROUND & AIMS: Hepatitis C virus (HCV) poses a global health problem, with over 170 million chronically infected individuals at risk of developing progressive liver disease. The ability of a virus to spread within a host is a key determinant of its persistence and virulence. HCV can transmit in vitro by cell-free particle diffusion or via contact(s) between infected and naïve hepatocytes. However, limited information is available on the relative efficiency of these routes, our aim is to develop physiologically relevant assays to quantify these processes. METHODS: We developed a single-cycle infection assay to measure HCV transmission rates. RESULTS: We compared HCV spread in proliferating and arrested cell systems and demonstrated a significant reduction in cell-to-cell infection of arrested target cells. Comparison of cell-free and cell-to-cell virus spread demonstrated relatively poor transmission rates, with 10-50 infected producer cells required to infect a single naïve target cell. We found HCV strain J6/JFH to be 10-fold more efficient at spreading via the cell-to-cell route than cell-free, whereas SA13/JFH and HK6/JFH strains showed comparable rates of infection via both routes. Importantly, the level of infectious virus released from cells did not predict the ability of a virus to spread in vitro, highlighting the importance of studying cell-associated viruses. CONCLUSIONS: These studies demonstrate the relatively poor infectivity of HCV and highlight differences between strains in their efficiency and preferred route of transmission that may inform future therapeutic strategies that target virus entry.

Zona L, Lupberger J, Sidahmed-Adrar N, Thumann C, Harris HJ, Barnes A, Florentin J, Tawar RG, Xiao F, Turek M et al. 2013. HRas signal transduction promotes hepatitis C virus cell entry by triggering assembly of the host tetraspanin receptor complex. Cell Host Microbe, 13 (3), pp. 302-313. | Show Abstract | Read more

Hepatitis C virus (HCV) entry is dependent on coreceptor complex formation between the tetraspanin superfamily member CD81 and the tight junction protein claudin-1 (CLDN1) on the host cell membrane. The receptor tyrosine kinase EGFR acts as a cofactor for HCV entry by promoting CD81-CLDN1 complex formation via unknown mechanisms. We identify the GTPase HRas, activated downstream of EGFR signaling, as a key host signal transducer for EGFR-mediated HCV entry. Proteomic analysis revealed that HRas associates with tetraspanin CD81, CLDN1, and the previously unrecognized HCV entry cofactors integrin β1 and Ras-related protein Rap2B in hepatocyte membranes. HRas signaling is required for lateral membrane diffusion of CD81, which enables tetraspanin receptor complex assembly. HRas was also found to be relevant for entry of other viruses, including influenza. Our data demonstrate that viruses exploit HRas signaling for cellular entry by compartmentalization of entry factors and receptor trafficking.

Harris HJ, Clerte C, Farquhar MJ, Goodall M, Hu K, Rassam P, Dosset P, Wilson GK, Balfe P, Ijzendoorn SC et al. 2013. Hepatoma polarization limits CD81 and hepatitis C virus dynamics. Cell Microbiol, 15 (3), pp. 430-445. | Show Abstract | Read more

Many viruses target the polarized epithelial apex during host invasion. In contrast, hepatitis C virus (HCV) engages receptors at the basal surface of hepatocytes in the polarized liver parenchyma. Hepatocyte polarization limits HCV entry by undefined mechanism(s). Given the recent reports highlighting a role for receptor mobility in pathogen entry, we studied the effect(s) of hepatocyte polarization on viral receptor and HCV pseudoparticle (HCVpp) dynamics using real-time fluorescence recovery after photobleaching and single particle tracking. Hepatoma polarization reduced CD81 and HCVpp dynamics at the basal membrane. Since cell polarization is accompanied by changes in the actin cytoskeleton and CD81 links to actin via its C-terminus, we studied the dynamics of a mutant CD81 lacking a C-terminal tail (CD81(ΔC)) and its effect(s) on HCVpp mobility and infection. CD81(ΔC) showed an increased frequency of confined trajectories and a reduction of Brownian diffusing molecules compared to wild-type protein in non-polarized cells. However, these changes were notobserved in polarized cells. HCVpp showed a significant reduction in Brownian diffusion and infection of CD81(ΔC) expressing non-polarized cells. In summary, these data highlight the dynamic nature of CD81 and demonstrate a role for CD81 lateral diffusion to regulate HCV infection in a polarization-dependent manner.

Harris HJ, Wilson GK, Hübscher SG, McKeating JA. 2013. Heterogeneous claudin-1 expression in human liver. Hepatology, 57 (2), pp. 854-855. | Read more

Wilkins C, Woodward J, Lau DT-Y, Barnes A, Joyce M, McFarlane N, McKeating JA, Tyrrell DL, Gale M. 2013. IFITM1 is a tight junction protein that inhibits hepatitis C virus entry. Hepatology, 57 (2), pp. 461-469. | Show Abstract | Read more

UNLABELLED: Type 1 interferon (IFN) continues to be the foundation for the current standard of care combination therapy for chronic hepatitis C virus (HCV) infection, yet the component interferon-stimulated genes (ISGs) that mediate the antiviral actions of IFN are not fully defined. Interferon-induced transmembrane protein 1 (IFITM1) is an ISG product that suppresses early stage infection by a number of viruses through an unknown mechanism of action. Moreover, the actions of IFITM1 on HCV infection are not fully elucidated. Here we identify IFITM1 as a hepatocyte tight junction protein and a potent anti-HCV effector molecule. IFITM1 expression is induced early during IFN treatment of hepatocytes and accumulates at hepatic tight junctions in HCV-infected human patient liver during IFN therapy. Additionally, we found that IFITM1 interacts with HCV coreceptors, including CD81 and occludin, to disrupt the process of viral entry. Thus, IFITM1 is an anti-HCV ISG whose actions impart control of HCV infection through interruption of viral coreceptor function. CONCLUSION: This study defines IFITM1 as an ISG effector with action against HCV entry. Design of therapy regimens to enhance IFITM1 expression should improve the virologic response among HCV patients undergoing treatment with type I IFN.

Bonander N, Jamshad M, Oberthür D, Clare M, Barwell J, Hu K, Farquhar MJ, Stamataki Z, Harris HJ, Dierks K et al. 2013. Production, purification and characterization of recombinant, full-length human claudin-1. PLoS One, 8 (5), pp. e64517. | Show Abstract | Read more

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

Davis C, Harris HJ, Hu K, Drummer HE, McKeating JA, Mullins JGL, Balfe P. 2012. In silico directed mutagenesis identifies the CD81/claudin-1 hepatitis C virus receptor interface. Cell Microbiol, 14 (12), pp. 1892-1903. | Show Abstract | Read more

Hepatitis C virus (HCV) entry is dependent on host cell molecules tetraspanin CD81, scavenger receptor BI and tight junction proteins claudin-1 and occludin. We previously reported a role for CD81/claudin-1 receptor complexes in HCV entry; however, the molecular mechanism(s) driving association between the receptors is unknown. We explored the molecular interface between CD81 and claudin-1 using a combination of bioinformatic sequence-based modelling, site-directed mutagenesis and Fluorescent Resonance Energy Transfer (FRET) imaging methodologies. Structural modelling predicts the first extracellular loop of claudin-1 to have a flexible beta conformation and identifies a motif between amino acids 62-66 that interacts with CD81 residues T149, E152 and T153. FRET studies confirm a role for these CD81 residues in claudin-1 association and HCV infection. Importantly, mutation of these CD81 residues has minimal impact on protein conformation or HCV glycoprotein binding, highlighting a new functional domain of CD81 that is essential for virus entry.

Brown RJP, Hudson N, Wilson G, Rehman SU, Jabbari S, Hu K, Tarr AW, Borrow P, Joyce M, Lewis J et al. 2012. Hepatitis C virus envelope glycoprotein fitness defines virus population composition following transmission to a new host. J Virol, 86 (22), pp. 11956-11966. | Show Abstract | Read more

Genetic variability is a hallmark of RNA virus populations. However, transmission to a new host often results in a marked decrease in population diversity. This genetic bottlenecking is observed during hepatitis C virus (HCV) transmission and can arise via a selective sweep or through the founder effect. To model HCV transmission, we utilized chimeric SCID/Alb-uPA mice with transplanted human hepatocytes and infected them with a human serum HCV inoculum. E1E2 glycoprotein gene sequences in the donor inoculum and recipient mice were determined following single-genome amplification (SGA). In independent experiments, using mice with liver cells grafted from different sources, an E1E2 variant undetectable in the source inoculum was selected for during transmission. Bayesian coalescent analyses indicated that this variant arose in the inoculum pretransmission. Transmitted variants that established initial infection harbored key substitutions in E1E2 outside HVR1. Notably, all posttransmission E1E2s had lost a potential N-linked glycosylation site (PNGS) in E2. In lentiviral pseudoparticle assays, the major posttransmission E1E2 variant conferred an increased capacity for entry compared to the major variant present in the inoculum. Together, these data demonstrate that increased envelope glycoprotein fitness can drive selective outgrowth of minor variants posttransmission and that loss of a PNGS is integral to this improved phenotype. Mathematical modeling of the dynamics of competing HCV variants indicated that relatively modest differences in glycoprotein fitness can result in marked shifts in virus population composition. Overall, these data provide important insights into the dynamics and selection of HCV populations during transmission.

Meredith LW, Wilson GK, Fletcher NF, McKeating JA. 2012. Hepatitis C virus entry: beyond receptors. Rev Med Virol, 22 (3), pp. 182-193. | Show Abstract | Read more

HCV is a blood-borne pathogen that affects approximately 3% of the global population and leads to progressive liver disease. Recent advances have identified an essential role for host cell molecules: tetraspanin CD81, scavenger receptor B1 and the tight junction proteins claudin-1 and occludin in HCV entry, suggesting a complex multi-step process. The conserved nature of this receptor-dependent step in the viral life cycle offers an attractive target for therapeutic intervention. Evidence is emerging that additional factors other than classical receptors, such as inflammatory mediators regulate the ability of hepatocytes to support HCV entry, and as such may provide potential avenues for drug design and development. In this review, we summarise the recent literature on HCV entry mechanisms with a view to realising the future potential of therapeutically targeting this process.

Fletcher NF, McKeating JA. 2012. Hepatitis C virus and the brain. J Viral Hepat, 19 (5), pp. 301-306. | Show Abstract | Read more

Hepatitis C virus (HCV) is an enveloped, positive-strand RNA virus of the family Flaviviridae that primarily infects hepatocytes, causing acute and chronic liver disease. HCV is also associated with a variety of extrahepatic symptoms including central nervous system (CNS) abnormalities, cognitive dysfunction, fatigue and depression. These symptoms do not correlate with the severity of liver disease and are independent of hepatic encephalopathy. HCV RNA has been associated with CNS tissue, and reports of viral sequence diversity between brain and liver tissue suggest independent viral evolution in the CNS and liver. This review will explore the data supporting HCV infection of the CNS and how this fits into our current understanding of HCV pathogenesis.

Farquhar MJ, Hu K, Harris HJ, Davis C, Brimacombe CL, Fletcher SJ, Baumert TF, Rappoport JZ, Balfe P, McKeating JA. 2012. Hepatitis C virus induces CD81 and claudin-1 endocytosis. J Virol, 86 (8), pp. 4305-4316. | Show Abstract | Read more

Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective, and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I, and the tight-junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. We demonstrate here that anti-CD81 antibodies inhibit HCV infection at late times after virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domains; however, CD81 lacks such motifs, leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin- and dynamin-dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 coendocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor-specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model wherein HCV stimulates receptor trafficking to promote particle internalization.

Wilson GK, Brimacombe CL, Rowe IA, Reynolds GM, Fletcher NF, Stamataki Z, Bhogal RH, Simões ML, Ashcroft M, Afford SC et al. 2012. A dual role for hypoxia inducible factor-1α in the hepatitis C virus lifecycle and hepatoma migration. J Hepatol, 56 (4), pp. 803-809. | Show Abstract | Read more

BACKGROUND & AIMS: Hepatitis C virus (HCV) causes progressive liver disease and is a major risk factor for the development of hepatocellular carcinoma (HCC). However, the role of infection in HCC pathogenesis is poorly understood. We investigated the effect(s) of HCV infection and viral glycoprotein expression on hepatoma biology to gain insights into the development of HCV associated HCC. METHODS: We assessed the effect(s) of HCV and viral glycoprotein expression on hepatoma polarity, migration and invasion. RESULTS: HCV glycoproteins perturb tight and adherens junction protein expression, and increase hepatoma migration and expression of epithelial to mesenchymal transition markers Snail and Twist via stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α regulates many genes involved in tumor growth and metastasis, including vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β). Neutralization of both growth factors shows different roles for VEGF and TGFβ in regulating hepatoma polarity and migration, respectively. Importantly, we confirmed these observations in virus infected hepatoma and primary human hepatocytes. Inhibition of HIF-1α reversed the effect(s) of infection and glycoprotein expression on hepatoma permeability and migration and significantly reduced HCV replication, demonstrating a dual role for HIF-1α in the cellular processes that are deregulated in many human cancers and in the viral life cycle. CONCLUSIONS: These data provide new insights into the cancer-promoting effects of HCV infection on HCC migration and offer new approaches for treatment.

Fletcher NF, Howard C, McKeating JA. 2012. Over the fence or through the gate: how viruses infect polarized cells. Immunotherapy, 4 (3), pp. 249-251. | Read more

Tarr AW, Urbanowicz RA, Jayaraj D, Brown RJP, McKeating JA, Irving WL, Ball JK. 2012. Naturally occurring antibodies that recognize linear epitopes in the amino terminus of the hepatitis C virus E2 protein confer noninterfering, additive neutralization. J Virol, 86 (5), pp. 2739-2749. | Show Abstract | Read more

Chronic hepatitis C virus (HCV) infection can persist even in the presence of a broadly neutralizing antibody response. Various mechanisms that underpin viral persistence have been proposed, and one of the most recently proposed mechanisms is the presence of interfering antibodies that negate neutralizing responses. Specifically, it has been proposed that antibodies targeting broadly neutralizing epitopes located within a region of E2 encompassing residues 412 to 423 can be inhibited by nonneutralizing antibodies binding to a less conserved region encompassing residues 434 to 446. To investigate this phenomenon, we characterized the neutralizing and inhibitory effects of human-derived affinity-purified immunoglobulin fractions and murine monoclonal antibodies and show that antibodies to both regions neutralize HCV pseudoparticle (HCVpp) and cell culture-infectious virus (HCVcc) infection albeit with different breadths and potencies. Epitope mapping revealed the presence of overlapping but distinct epitopes in both regions, which may explain the observed differences in neutralizing phenotypes. Crucially, we failed to demonstrate any inhibition between these two groups of antibodies, suggesting that interference by nonneutralizing antibodies, at least for the region encompassing residues 434 to 446, does not provide a mechanism for HCV persistence in chronically infected individuals.

Fletcher NF, Wilson GK, Murray J, Hu K, Lewis A, Reynolds GM, Stamataki Z, Meredith LW, Rowe IA, Luo G et al. 2012. Hepatitis C virus infects the endothelial cells of the blood-brain barrier. Gastroenterology, 142 (3), pp. 634-643.e6. | Show Abstract | Read more

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS. METHODS: We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells by using quantitative polymerase chain reaction and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture-derived HCV were used to study the ability of endothelial cells to support viral entry and replication. RESULTS: Using quantitative polymerase chain reaction, we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hCMEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor BI, and claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis. CONCLUSIONS: Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies.

Howl J, Matou-Nasri S, West DC, Farquhar M, Slaninová J, Ostenson C-G, Zorko M, Ostlund P, Kumar S, Langel U et al. 2012. Bioportide: an emergent concept of bioactive cell-penetrating peptides. Cell Mol Life Sci, 69 (17), pp. 2951-2966. | Show Abstract | Read more

Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents.

Stamataki Z, Coates S, Abrignani S, Houghton M, McKeating JA. 2011. Immunization of human volunteers with hepatitis C virus envelope glycoproteins elicits antibodies that cross-neutralize heterologous virus strains. J Infect Dis, 204 (5), pp. 811-813. | Read more

Petrovic D, Stamataki Z, Dempsey E, Golden-Mason L, Freeley M, Doherty D, Prichard D, Keogh C, Conroy J, Mitchell S et al. 2011. Hepatitis C virus targets the T cell secretory machinery as a mechanism of immune evasion. Hepatology, 53 (6), pp. 1846-1853. | Show Abstract | Read more

UNLABELLED: T cell activation and the resultant production of interleukin (IL-2) is a central response of the adaptive immune system to pathogens, such as hepatitis C virus (HCV). HCV uses several mechanisms to evade both the innate and adaptive arms of the immune response. Here we demonstrate that liver biopsy specimens from individuals infected with HCV had significantly lower levels of IL-2 compared with those with other inflammatory liver diseases. Cell culture-grown HCV particles inhibited the production of IL-2 by normal peripheral blood mononuclear cells, as did serum from HCV-infected patients. This process was mediated by the interaction of HCV envelope protein E2 with tetraspanin CD81 coreceptor. HCV E2 attenuated IL-2 production at the level of secretion and not transcription by targeting the translocation of protein kinase C beta (PKCβ), which is essential for IL-2 secretion, to lipid raft microdomains. The lipid raft disruptor methyl-β-cyclodextrin reversed HCV E2-mediated inhibition of IL-2 secretion, but not in the presence of a PKCβ-selective inhibitor. HCV E2 further inhibited the secretion of other cytokines, including interferon-γ. CONCLUSION: These data suggest that HCV E2-mediated disruption of the association of PKCβ with the cellular secretory machinery represents a novel mechanism for HCV to evade the human immune response and to establish persistent infection.

Lupberger J, Zeisel MB, Xiao F, Thumann C, Fofana I, Zona L, Davis C, Mee CJ, Turek M, Gorke S et al. 2011. EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nat Med, 17 (5), pp. 589-595. | Show Abstract | Read more

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.

Farquhar MJ, Harris HJ, McKeating JA. 2011. Hepatitis C virus entry and the tetraspanin CD81. Biochem Soc Trans, 39 (2), pp. 532-536. | Show Abstract | Read more

CD81, a member of the tetraspanin integral membrane protein family, has been identified as an essential receptor for HCV (hepatitis C virus). The present review highlights recent published data on the role that CD81 plays in HCV entry, including the importance of actin-dependent lateral diffusion of CD81 within the cell membrane, CD81 endocytosis and the CD81-Claudin-1 receptor complex in HCV internalization. Additional functions for CD81 in the viral life cycle and the role of HCV-CD81 interactions in HCV-induced B-cell and CNS (central nervous system) abnormalities are discussed.

Bonander N, Jamshad M, Hu K, Farquhar MJ, Stamataki Z, Balfe P, McKeating JA, Bill RM. 2011. Structural characterization of CD81-Claudin-1 hepatitis C virus receptor complexes. Biochem Soc Trans, 39 (2), pp. 537-540. | Show Abstract | Read more

Tetraspanins are thought to exert their biological function(s) by co-ordinating the lateral movement and trafficking of associated molecules into tetraspanin-enriched microdomains. A second four-TM (transmembrane) domain protein family, the Claudin superfamily, is the major structural component of cellular TJs (tight junctions). Although the Claudin family displays low sequence homology and appears to be evolutionarily distinct from the tetraspanins, CD81 and Claudin-1 are critical molecules defining HCV (hepatitis C virus) entry; we recently demonstrated that CD81-Claudin-1 complexes have an essential role in this process. To understand the molecular basis of CD81-Claudin-1 complex formation, we produced and purified milligram quantities of full-length CD81 and Claudin-1, alone and in complex, in both detergent and lipid contexts. Structural characterization of these purified proteins will allow us to define the mechanism(s) underlying virus-cell interactions and aid the design of therapeutic agents targeting early steps in the viral life cycle.

Syder AJ, Lee H, Zeisel MB, Grove J, Soulier E, Macdonald J, Chow S, Chang J, Baumert TF, McKeating JA et al. 2011. Small molecule scavenger receptor BI antagonists are potent HCV entry inhibitors. J Hepatol, 54 (1), pp. 48-55. | Show Abstract | Read more

BACKGROUND AND AIMS: ITX 5061 is a clinical stage small molecule compound that promotes high-density lipoprotein (HDL) levels in animals and patients by targeting the scavenger receptor BI protein pathway. Since SR-BI is a known co-receptor for HCV infection, we evaluated these compounds for their effects on HCV entry. METHODS: We obtained ITX 5061 and related compounds to characterize their interaction with SR-BI and effects on HCV entry and infection. RESULTS: We confirmed that a tritium-labeled compound analog (ITX 7650) binds cells expressing SR-BI, and both ITX 5061 and ITX 7650 compete for HDL-mediated lipid transfer in an SR-BI dependent manner. Both molecules inhibit HCVcc and HCVpp infection of primary human hepatocytes and/or human hepatoma cell lines and have minimal effects on HCV RNA replication. Kinetic studies suggest that the compounds act at an early post-binding step. CONCLUSIONS: These results suggest that the ITX compounds inhibit HCV infection with a mechanism of action distinct from other HCV therapies under development. Since ITX 5061 has already been evaluated in over 280 patients with good pharmacokinetic and safety profiles, it warrants proof-of-concept clinical studies in HCV infected patients.

Stamataki Z, Tilakaratne S, Adams DH, McKeating JA. 2011. Rituximab treatment in hepatitis C infection: an in vitro model to study the impact of B cell depletion on virus infectivity. PLoS One, 6 (9), pp. e25789. | Show Abstract | Read more

Hepatitis C virus (HCV) infected patients with vasculitis are often treated with the B-cell-depleting anti-CD20 antibody rituximab. Treatment reduces the cryoglobulins that cause vasculitis, yet it also leads to a transient increase in liver enzymes and HCV genomic RNA in the periphery. The mechanism underlying the increased viral load is unclear and both direct and indirect roles have been proposed for B cells in HCV infection. We previously reported that HCV can associate with B cells and can trans-infect hepatocytes. We established an in vitro assay to study the effect(s) of rituximab on B cell-associated HCV infectivity. Rituximab-mediated lysis of B cells in vitro increases the level of infectious HCV released from B cells. Our results, using a model where virus does not replicate in B cells, recapitulate observations seen in patients and may explain in part the rapid increase in blood HCV RNA observed after rituximab treatment.

Brimacombe CL, Grove J, Meredith LW, Hu K, Syder AJ, Flores MV, Timpe JM, Krieger SE, Baumert TF, Tellinghuisen TL et al. 2011. Neutralizing antibody-resistant hepatitis C virus cell-to-cell transmission. J Virol, 85 (1), pp. 596-605. | Show Abstract | Read more

Hepatitis C virus (HCV) can initiate infection by cell-free particle and cell-cell contact-dependent transmission. In this study we use a novel infectious coculture system to examine these alternative modes of infection. Cell-to-cell transmission is relatively resistant to anti-HCV glycoprotein monoclonal antibodies and polyclonal immunoglobulin isolated from infected individuals, providing an effective strategy for escaping host humoral immune responses. Chimeric viruses expressing the structural proteins representing the seven major HCV genotypes demonstrate neutralizing antibody-resistant cell-to-cell transmission. HCV entry is a multistep process involving numerous receptors. In this study we demonstrate that, in contrast to earlier reports, CD81 and the tight-junction components claudin-1 and occludin are all essential for both cell-free and cell-to-cell viral transmission. However, scavenger receptor BI (SR-BI) has a more prominent role in cell-to-cell transmission of the virus, with SR-BI-specific antibodies and small-molecule inhibitors showing preferential inhibition of this infection route. These observations highlight the importance of targeting host cell receptors, in particular SR-BI, to control viral infection and spread in the liver.

Fletcher NF, Yang JP, Farquhar MJ, Hu K, Davis C, He Q, Dowd K, Ray SC, Krieger SE, Neyts J et al. 2010. Hepatitis C virus infection of neuroepithelioma cell lines. Gastroenterology, 139 (4), pp. 1365-1374. | Show Abstract | Read more

BACKGROUND & AIMS: Hepatitis C virus (HCV) establishes chronic infections in 3% of the world's population. Infection leads to progressive liver disease; hepatocytes are the major site of viral replication in vivo. However, chronic infection is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. We therefore screened a series of neural and brain-derived cell lines for their ability to support HCV entry and replication. METHODS: We used a panel of neural-derived cell lines, HCV pseudoparticles (HCVpp), and an infectious, HCV JFH-1 cell-culture system (HCVcc) to assess viral tropism. RESULTS: Two independently derived neuroepithelioma cell lines (SK-N-MC and SK-PN-DW) permitted HCVpp entry. In contrast, several neuroblastoma, glioma, and astrocytoma cell lines were refractory to HCVpp infection. HCVcc infected the neuroepithelioma cell lines and established a productive infection. Permissive neuroepithelioma cells expressed CD81, scavenger receptor BI (SR-BI), and the tight junction proteins Claudin-1 (CLDN1) and occludin, whereas nonpermissive neural cell lines lacked CLDN1 and, in some cases, SR-BI. HCVpp infection of the neuroepithelioma cells was neutralized by antibodies to CD81, SR-BI, CLDN1, and HCV E2. Furthermore, anti-CD81, interferon, and the anti-NS3 protease inhibitor VX-950 significantly reduced HCVcc infection of neuroepithelioma and hepatoma cells. CONCLUSIONS: Neuroepithelioma-derived cell lines express functional receptors that support HCV entry at levels comparable to those of hepatoma cells. HCV infection in vitro is not restricted to hepatic-derived cells, so HCV might infect cells of the CNS in vivo.

Fofana I, Krieger SE, Grunert F, Glauben S, Xiao F, Fafi-Kremer S, Soulier E, Royer C, Thumann C, Mee CJ et al. 2010. Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes. Gastroenterology, 139 (3), pp. 953-964.e4. | Show Abstract | Read more

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies. The tight junction protein claudin-1 (CLDN1) has been shown to be required for entry of HCV into the cell. METHODS: Using genetic immunization, we produced 6 monoclonal antibodies against the host entry factor CLDN1. The effects of antibodies on HCV infection were analyzed in human cell lines and primary human hepatocytes. RESULTS: Competition and binding studies demonstrated that antibodies interacted with conformational epitopes of the first extracellular loop of CLDN1; binding of these antibodies required the motif W(30)-GLW(51)-C(54)-C(64) and residues in the N-terminal third of CLDN1. The monoclonal antibodies against CLDN1 efficiently inhibited infection by HCV of all major genotypes as well as highly variable HCV quasispecies isolated from individual patients. Furthermore, antibodies efficiently blocked cell entry of highly infectious escape variants of HCV that were resistant to neutralizing antibodies. CONCLUSIONS: Monoclonal antibodies against the HCV entry factor CLDN1 might be used to prevent HCV infection, such as after liver transplantation, and might also restrain virus spread in chronically infected patients.

Thorley JA, McKeating JA, Rappoport JZ. 2010. Mechanisms of viral entry: sneaking in the front door. Protoplasma, 244 (1-4), pp. 15-24. | Show Abstract | Read more

Recent developments in methods to study virus internalisation are providing clearer insights into mechanisms used by viruses to enter host cells. The use of dominant negative constructs, specific inhibitory drugs and RNAi to selectively prevent entry through particular pathways has provided evidence for the clathrin-mediated entry of hepatitis C virus (HCV) as well as the caveolar entry of Simian Virus 40. Moreover, the ability to image and track fluorescent-labelled virus particles in real-time has begun to challenge the classical plasma membrane entry mechanisms described for poliovirus and human immunodeficiency virus. This review will cover both well-documented entry mechanisms as well as more recent discoveries in the entry pathways of enveloped and non-enveloped viruses. This will include viruses which enter the cytosol directly at the plasma membrane and those which enter via endocytosis and traversal of internal membrane barrier(s). Recent developments in imaging and inhibition of entry pathways have provided insights into the ill-defined entry mechanism of HCV, bringing it to the forefront of viral entry research. Finally, as high-affinity receptors often define viral internalisation pathways, and tropism in vivo, host membrane proteins to which viral particles specifically bind will be discussed throughout.

Harris HJ, Davis C, Mullins JGL, Hu K, Goodall M, Farquhar MJ, Mee CJ, McCaffrey K, Young S, Drummer H et al. 2010. Claudin association with CD81 defines hepatitis C virus entry. J Biol Chem, 285 (27), pp. 21092-21102. | Show Abstract | Read more

Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.

Wagoner J, Negash A, Kane OJ, Martinez LE, Nahmias Y, Bourne N, Owen DM, Grove J, Brimacombe C, McKeating JA et al. 2010. Multiple effects of silymarin on the hepatitis C virus lifecycle. Hepatology, 51 (6), pp. 1912-1921. | Show Abstract | Read more

UNLABELLED: Silymarin, an extract from milk thistle (Silybum marianum), and its purified flavonolignans have been recently shown to inhibit hepatitis C virus (HCV) infection, both in vitro and in vivo. In the current study, we further characterized silymarin's antiviral actions. Silymarin had antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin but not silibinin inhibited genotype 2a NS5B RNA-dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti-HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b BK and four 1b RDRPs derived from HCV-infected patients. Moreover, silymarin did not inhibit HCV replication in five independent genotype 1a, 1b, and 2a replicon cell lines that did not produce infectious virus. Silymarin inhibited microsomal triglyceride transfer protein activity, apolipoprotein B secretion, and infectious virion production into culture supernatants. Silymarin also blocked cell-to-cell spread of virus. CONCLUSION: Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarin's antiviral action appear to include blocking of virus entry and transmission, possibly by targeting the host cell.

Lemon SM, McKeating JA, Pietschmann T, Frick DN, Glenn JS, Tellinghuisen TL, Symons J, Furman PA. 2010. Development of novel therapies for hepatitis C. Antiviral Res, 86 (1), pp. 79-92. | Show Abstract | Read more

The current standard of care for the treatment of hepatitis C virus (HCV) infection is a combination of pegylated IFN and ribavirin (Peg-IFN/RBV). Because of the adverse effects associated with both IFN and ribavirin and because Peg-IFN/RBV provides only about a 45-50% sustained virological response (SVR, undetectable HCV RNA for greater than 24 weeks after cessation of therapy) in genotype 1-infected individuals, there is a need for more potent anti-HCV compounds with fewer adverse effects. The twenty-first International Conference on Antiviral Research held in May 2009 in Miami Beach, Florida, featured a special session focused on novel targets for HCV therapy. The session included presentations by world-renowned experts in HCV virology and covered a diverse array of potential targets for the development of new classes of HCV therapies. This review contains concise summaries of discussed topics that included the innate immune response, virus entry, the NS2 protease, the NS3 helicase, NS4B, and NS5A. Each presenter discussed the current knowledge of these targets and provided examples of recent scientific breakthroughs that are enhancing our understanding of these targets. As our understanding of the role of these novel anti-HCV targets increases so will our ability to discover new, more safe and effective anti-HCV therapies.

Krieger SE, Zeisel MB, Davis C, Thumann C, Harris HJ, Schnober EK, Mee C, Soulier E, Royer C, Lambotin M et al. 2010. Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2-CD81-claudin-1 associations. Hepatology, 51 (4), pp. 1144-1157. | Show Abstract | Read more

UNLABELLED: The tight junction protein claudin-1 (CLDN1) has been shown to be essential for hepatitis C virus (HCV) entry-the first step of viral infection. Due to the lack of neutralizing anti-CLDN1 antibodies, the role of CLDN1 in the viral entry process is poorly understood. In this study, we produced antibodies directed against the human CLDN1 extracellular loops by genetic immunization and used these antibodies to investigate the mechanistic role of CLDN1 for HCV entry in an infectious HCV cell culture system and human hepatocytes. Antibodies specific for cell surface-expressed CLDN1 specifically inhibit HCV infection in a dose-dependent manner. Antibodies specific for CLDN1, scavenger receptor B1, and CD81 show an additive neutralizing capacity compared with either agent used alone. Kinetic studies with anti-CLDN1 and anti-CD81 antibodies demonstrate that HCV interactions with both entry factors occur at a similar time in the internalization process. Anti-CLDN1 antibodies inhibit the binding of envelope glycoprotein E2 to HCV permissive cell lines in the absence of detectable CLDN1-E2 interaction. Using fluorescent-labeled entry factors and fluorescence resonance energy transfer methodology, we demonstrate that anti-CLDN1 antibodies inhibit CD81-CLDN1 association. In contrast, CLDN1-CLDN1 and CD81-CD81 associations were not modulated. Taken together, our results demonstrate that antibodies targeting CLDN1 neutralize HCV infectivity by reducing E2 association with the cell surface and disrupting CD81-CLDN1 interactions. CONCLUSION: These results further define the function of CLDN1 in the HCV entry process and highlight new antiviral strategies targeting E2-CD81-CLDN1 interactions.

Mee CJ, Farquhar MJ, Harris HJ, Hu K, Ramma W, Ahmed A, Maurel P, Bicknell R, Balfe P, McKeating JA. 2010. Hepatitis C virus infection reduces hepatocellular polarity in a vascular endothelial growth factor-dependent manner. Gastroenterology, 138 (3), pp. 1134-1142. | Show Abstract | Read more

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection leads to progressive liver disease, frequently culminating in fibrosis and hepatocellular carcinoma. The mechanisms underlying liver injury in chronic hepatitis C are poorly understood. This study evaluated the role of vascular endothelial growth factor (VEGF) in hepatocyte polarity and HCV infection. METHODS: We used polarized hepatoma cell lines and the recently described infectious HCV Japanese fulminant hepatitis (JFH)-1 cell culture system to study the role of VEGF in regulating hepatoma permeability and HCV infection. RESULTS: VEGF negatively regulates hepatocellular tight junction integrity and cell polarity by a novel VEGF receptor 2-dependent pathway. VEGF reduced hepatoma tight junction integrity, induced a re-organization of occludin, and promoted HCV entry. Conversely, inhibition of hepatoma expressed VEGF with the receptor kinase inhibitor sorafenib or with neutralizing anti-VEGF antibodies promoted polarization and inhibited HCV entry, showing an autocrine pathway. HCV infection of primary hepatocytes or hepatoma cell lines promoted VEGF expression and reduced their polarity. Importantly, treatment of HCV-infected cells with VEGF inhibitors restored their ability to polarize, showing a VEGF-dependent pathway. CONCLUSIONS: Hepatic polarity is critical to normal liver physiology. HCV infection promotes VEGF expression that depolarizes hepatoma cells, promoting viral transmission and lymphocyte migration into the parenchyma that may promote hepatocyte injury.

Schwarz AK, Grove J, Hu K, Mee CJ, Balfe P, McKeating JA. 2009. Hepatoma cell density promotes claudin-1 and scavenger receptor BI expression and hepatitis C virus internalization. J Virol, 83 (23), pp. 12407-12414. | Show Abstract | Read more

Hepatitis C virus (HCV) entry occurs via a pH- and clathrin-dependent endocytic pathway and requires a number of cellular factors, including CD81, the tight-junction proteins claudin 1 (CLDN1) and occludin, and scavenger receptor class B member I (SR-BI). HCV tropism is restricted to the liver, where hepatocytes are tightly packed. Here, we demonstrate that SR-BI and CLDN1 expression is modulated in confluent human hepatoma cells, with both receptors being enriched at cell-cell junctions. Cellular contact increased HCV pseudoparticle (HCVpp) and HCV particle (HCVcc) infection and accelerated the internalization of cell-bound HCVcc, suggesting that the cell contact modulation of receptor levels may facilitate the assembly of receptor complexes required for virus internalization. CLDN1 overexpression in subconfluent cells was unable to recapitulate this effect, whereas increased SR-BI expression enhanced HCVpp entry and HCVcc internalization, demonstrating a rate-limiting role for SR-BI in HCV internalization.

Balfe P, McKeating JA. 2009. The complexities of hepatitis C virus entry. J Hepatol, 51 (3), pp. 609-611. | Show Abstract | Read more

Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Ploss A, Evans MJ, Gaysinskaya VA, Panis M, You H, de Jong YP, Rice CM. Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. The development of much needed specific antiviral therapies and an effective vaccine has been hampered by the lack of a convenient small animal model. The determinants restricting HCV tropism to human and chimpanzee hosts are unknown. Replication of the viral RNA has been demonstrated in mouse cells, but these cells are not infectable with either lentiviral particles bearing HCV glycoproteins (HCVpp) or HCV produced in cell culture (HCVcc) (A.P., M.E. and C.M.R., unpublished observations), suggesting that there is a block at the level of entry. Here we show, using an iterative complementary DNA library screening approach, that human occludin (OCLN) is an essential HCV cell entry factor that is able to render murine cells infectable with HCVpp. Similarly, OCLN is required for the HCV-susceptibility of human cells, because its overexpression in uninfectable cells specifically enhanced HCVpp uptake, whereas its silencing in permissive cells impaired both HCVpp and HCVcc infection. In addition to OCLN, HCVpp infection of murine cells required expression of the previously identified HCV entry factors CD81 (ref. 4), scavenger receptor class B type I (SR-BI, also known as SCARB1) and claudin-1 (CLDN1). Although the mouse versions of SR-BI and CLDN1 function at least as well as the human proteins in promoting HCV entry, both OCLN and CD81 must be of human origin to allow efficient infection. The species-specific determinants of OCLN were mapped to its second extracellular loop. The identification of OCLN as a new HCV entry factor further highlights the importance of the tight junction complex in the viral entry process, and provides an important advance towards efforts to develop small animal models for HCV. [Abstract reproduced by permission of Nature 2009;457:882-886]. © 2009.

Keck Z-Y, Li SH, Xia J, von Hahn T, Balfe P, McKeating JA, Witteveldt J, Patel AH, Alter H, Rice CM, Foung SKH. 2009. Mutations in hepatitis C virus E2 located outside the CD81 binding sites lead to escape from broadly neutralizing antibodies but compromise virus infectivity. J Virol, 83 (12), pp. 6149-6160. | Show Abstract | Read more

Broadly neutralizing antibodies are commonly present in the sera of patients with chronic hepatitis C virus (HCV) infection. To elucidate possible mechanisms of virus escape from these antibodies, retrovirus particles pseudotyped with HCV glycoproteins (HCVpp) isolated from sequential samples collected over a 26-year period from a chronically infected patient, H, were used to characterize the neutralization potential and binding affinity of a panel of anti-HCV E2 human monoclonal antibodies (HMAbs). Moreover, AP33, a neutralizing murine monoclonal antibody (MAb) to a linear epitope in E2, was also tested against selected variants. The HMAbs used were previously shown to broadly neutralize HCV and to recognize a cluster of highly immunogenic overlapping epitopes, designated domain B, containing residues that are also critical for binding of viral E2 glycoprotein to CD81, a receptor essential for virus entry. Escape variants were observed at different time points with some of the HMAbs. Other HMAbs neutralized all variants except for the isolate 02.E10, obtained in 2002, which was also resistant to MAb AP33. The 02.E10 HCVpp that have reduced binding affinities for all antibodies and for CD81 also showed reduced infectivity. Comparison of the 02.E10 nucleotide sequence with that of the strain H-derived consensus variant, H77c, revealed the former to have two mutations in E2, S501N and V506A, located outside the known CD81 binding sites. Substitution A506V in 02.E10 HCVpp restored binding to CD81, but its antibody neutralization sensitivity was only partially restored. Double substitutions comprising N501S and A506V synergistically restored 02.E10 HCVpp infectivity. Other mutations that are not part of the antibody binding epitope in the context of N501S and A506V were able to completely restore neutralization sensitivity. These findings showed that some nonlinear overlapping epitopes are more essential than others for viral fitness and consequently are more invariant during earlier years of chronic infection. Further, the ability of the 02.E10 consensus variant to escape neutralization by the tested antibodies could be a new mechanism of virus escape from immune containment. Mutations that are outside receptor binding sites resulted in structural changes leading to complete escape from domain B neutralizing antibodies, while simultaneously compromising viral fitness by reducing binding to CD81.

Mee CJ, Harris HJ, Farquhar MJ, Wilson G, Reynolds G, Davis C, van IJzendoorn SCD, Balfe P, McKeating JA. 2009. Polarization restricts hepatitis C virus entry into HepG2 hepatoma cells. J Virol, 83 (12), pp. 6211-6221. | Show Abstract | Read more

The primary reservoir for hepatitis C virus (HCV) replication is believed to be hepatocytes, which are highly polarized with tight junctions (TJ) separating their basolateral and apical domains. HepG2 cells develop polarity over time, resulting in the formation and remodeling of bile canalicular (BC) structures. HepG2 cells expressing CD81 provide a model system to study the effects of hepatic polarity on HCV infection. We found an inverse association between HepG2-CD81 polarization and HCV pseudoparticle entry. As HepG2 cells polarize, discrete pools of claudin-1 (CLDN1) at the TJ and basal/lateral membranes develop, consistent with the pattern of receptor staining observed in liver tissue. The TJ and nonjunctional pools of CLDN1 show an altered association with CD81 and localization in response to the PKA antagonist Rp-8-Br-cyclic AMPs (cAMPs). Rp-8-Br-cAMPs reduced CLDN1 expression at the basal membrane and inhibited HCV infection, supporting a model where the nonjunctional pools of CLDN1 have a role in HCV entry. Treatment of HepG2 cells with proinflammatory cytokines, tumor necrosis factor alpha and gamma interferon, perturbed TJ integrity but had minimal effect(s) on cellular polarity and HCV infection, suggesting that TJ integrity does not limit HCV entry into polarized HepG2 cells. In contrast, activation of PKC with phorbol ester reduced TJ integrity, ablated HepG2 polarity, and stimulated HCV entry. Overall, these data show that complex hepatocyte-like polarity alters CLDN1 localization and limits HCV entry, suggesting that agents which disrupt hepatocyte polarity may promote HCV infection and transmission within the liver.

Stamataki Z, Shannon-Lowe C, Shaw J, Mutimer D, Rickinson AB, Gordon J, Adams DH, Balfe P, McKeating JA. 2009. Hepatitis C virus association with peripheral blood B lymphocytes potentiates viral infection of liver-derived hepatoma cells. Blood, 113 (3), pp. 585-593. | Show Abstract | Read more

Hepatitis C virus (HCV) primarily replicates within the liver, leading to hepatitis, fibrosis, and hepatocellular carcinoma. Infection is also associated with B-cell abnormalities, suggesting an association of the virus with B cells. The infectious JFH-1 strain of HCV can bind primary and immortalized B cells but fails to establish productive infection. However, B cell-associated virus readily infects hepatoma cells, showing an enhanced infectivity compared with extracellular virus. B cells express the viral receptors CD81, SR-BI, and the C-type lectins DC-SIGN and L-SIGN. Antibodies specific for SR-BI and DC-SIGN/L-SIGN reduced B-cell transinfection, supporting a role for these molecules in B-cell association with HCV. Stimulation of B cells with CD40 ligand and interleukin-4 promoted their ability to transinfect hepatoma cells. B cell-associated virus is resistant to trypsin proteolysis and HCV-specific neutralizing antibodies, consistent with particle internalization. HCV promoted the adhesion of primary B cells to Huh-7 hepatomas, providing a mechanism for B-cell retention in the infected liver. In summary, B cells may provide a vehicle for HCV to persist and transmit to the liver.

Gal-Tanamy M, Keck Z-Y, Yi M, McKeating JA, Patel AH, Foung SKH, Lemon SM. 2008. In vitro selection of a neutralization-resistant hepatitis C virus escape mutant. Proc Natl Acad Sci U S A, 105 (49), pp. 19450-19455. | Show Abstract | Read more

Effective immunization against hepatitis C virus (HCV) infections is likely to require the induction of both robust T and B cell immunity. Although neutralizing antibodies may play an important role in control of infection, there is little understanding of the structure of the HCV envelope glycoproteins and how they interact with such antibodies. An additional challenge for vaccine design is the genetic diversity of HCV and the rapid evolution of viral quasispecies that escape antibody-mediated neutralization. We used a cell culture-infectious, chimeric HCV with the structural proteins of genotype 1a virus to identify envelope residues contributing to the epitope recognized by a broadly neutralizing, murine monoclonal antibody, AP33. By repetitive rounds of neutralization followed by amplification, we selected a population of viral escape mutants that resist stringent neutralization with AP33 and no longer bind the antibody. Two amino acid substitutions, widely separated in the linear sequence of the E2 envelope protein (N415Y and E655G), were identified by sequencing of cloned cDNA and shown by reverse genetics analysis to contribute jointly to the AP33 resistance phenotype. The N415Y mutation substantially lowered virus fitness, most likely because of a defect in viral entry, but did not reduce binding of soluble CD81 to immobilized HCV-pseudotyped retrovirus particles. The in vitro selection of an HCV escape mutant recapitulates the ongoing evolution of antigenic variants that contributes to viral persistence in humans and reveals information concerning the conformational structure of the AP33 epitope, its role in viral replication, and constraints on its molecular evolution.

Farquhar MJ, McKeating JA. 2008. Primary hepatocytes as targets for hepatitis C virus replication. J Viral Hepat, 15 (12), pp. 849-854. | Show Abstract | Read more

Much of our current understanding of hepatitis C virus (HCV) replication has hailed from the use of a small number of cloned viral genomes and transformed hepatoma cell lines. Recent evidence suggests that lipoproteins play a key role in the HCV life cycle and virus particles derived from the sera of infected patients exist in association with host lipoproteins. This report will review the literature on HCV replication in primary hepatocytes and transformed cell lines, focusing largely on host factors defining particle entry.

Timpe JM, McKeating JA. 2008. Hepatitis C virus entry: possible targets for therapy. Gut, 57 (12), pp. 1728-1737. | Read more

Grove J, Nielsen S, Zhong J, Bassendine MF, Drummer HE, Balfe P, McKeating JA. 2008. Identification of a residue in hepatitis C virus E2 glycoprotein that determines scavenger receptor BI and CD81 receptor dependency and sensitivity to neutralizing antibodies. J Virol, 82 (24), pp. 12020-12029. | Show Abstract | Read more

Hepatitis C virus (HCV) infection is dependent on at least three coreceptors: CD81, scavenger receptor BI (SR-BI), and claudin-1. The mechanism of how these molecules coordinate HCV entry is unknown. In this study we demonstrate that a cell culture-adapted JFH-1 mutant, with an amino acid change in E2 at position 451 (G451R), has a reduced dependency on SR-BI. This altered receptor dependency is accompanied by an increased sensitivity to neutralization by soluble CD81 and enhanced binding of recombinant E2 to cell surface-expressed and soluble CD81. Fractionation of HCV by density gradient centrifugation allows the analysis of particle-lipoprotein associations. The cell culture-adapted mutation alters the relationship between particle density and infectivity, with the peak infectivity occurring at higher density than the parental virus. No association was observed between particle density and SR-BI or CD81 coreceptor dependence. JFH-1 G451R is highly sensitive to neutralization by gp-specific antibodies, suggesting increased epitope exposure at the virion surface. Finally, an association was observed between JFH-1 particle density and sensitivity to neutralizing antibodies (NAbs), suggesting that lipoprotein association reduces the sensitivity of particles to NAbs. In summary, mutation of E2 at position 451 alters the relationship between particle density and infectivity, disrupts coreceptor dependence, and increases virion sensitivity to receptor mimics and NAbs. Our data suggest that a balanced interplay between HCV particles, lipoprotein components, and viral receptors allows the evasion of host immune responses.

Farquhar MJ, Harris HJ, Diskar M, Jones S, Mee CJ, Nielsen SU, Brimacombe CL, Molina S, Toms GL, Maurel P et al. 2008. Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity. J Virol, 82 (17), pp. 8797-8811. | Show Abstract | Read more

Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.

Stamataki Z, Grove J, Balfe P, McKeating JA. 2008. Hepatitis C virus entry and neutralization. Clin Liver Dis, 12 (3), pp. 693-x. | Show Abstract | Read more

The processes of hepatitis C virus (HCV) entry and antibody-mediated neutralization are intimately linked. The high frequency of neutralizing antibodies (nAbs) that inhibit E2-CD81 interaction(s) suggests that this is a major target for the humoral immune response. The observation that HCV can transmit to naive cells by means of CD81-dependent and -independent routes in vitro awaits further investigation to assess the significance in vivo but may offer new strategies for HCV to escape nAbs. The identification of claudins in the entry process highlights the importance of cell polarity in defining routes of HCV entry and release, with recent experiments suggesting a polarized route of viral entry into cells in vitro. In this review, the authors summarize the current understanding of the mechanism(s) defining HCV entry and the role of nAbs in controlling HCV replication.

Mizukoshi E, Eisenbach C, Edlin BR, Newton KP, Raghuraman S, Weiler-Normann C, Tobler LH, Busch MP, Carrington M, McKeating JA et al. 2008. Hepatitis C virus (HCV)-specific immune responses of long-term injection drug users frequently exposed to HCV. J Infect Dis, 198 (2), pp. 203-212. | Show Abstract | Read more

BACKGROUND: Injection drug users (IDUs) who successfully clear hepatitis C virus (HCV) have a reduced risk of developing chronic reinfection, despite their continuing exposure to the virus. To identify immunological correlates for this apparent protection, we studied HCV-specific immune responses in long-term IDUs (duration, >10 years). METHODS: HCV-specific T cell responses were assessed in proliferation, enzyme-linked immunospot (ELISPOT), interferon (IFN)-gamma secretion, and cytotoxicity assays, whereas HCV-specific antibodies were assessed in enzyme immunoassays (EIAs), chemiluminescent assays, and in vitro neutralization assays. RESULTS: HCV-specific T cell proliferation and IFN-gamma production were more common in nonviremic EIA-positive IDUs (16 [94%] of 17 IDUs) than in viremic EIA-positive IDUs (9 [45%] of 20 IDUs) (P= .003). They were also noted in 16 (62%) of 26 nonviremic EIA-negative IDUs. In contrast, 19 (90%) of 21 viremic IDUs displayed neutralizing antibodies (nAbs), compared with 9 (56%) of 16 nonviremic EIA-positive IDUs (P= .04) and 0 of 24 nonviremic EIA-negative IDUs. Nonviremic IDUs with nAbs were older (P= .0115) than those without nAbs, but these groups did not differ in terms of either injection drug use duration or HCV-specific T cell responses. CONCLUSION: The reduced risk of HCV persistence in IDUs previously recovered from HCV infection correlated with T cell responses, and prolonged antigenic stimulation appears to be required to maintain humoral responses.

Harris HJ, Farquhar MJ, Mee CJ, Davis C, Reynolds GM, Jennings A, Hu K, Yuan F, Deng H, Hubscher SG et al. 2008. CD81 and claudin 1 coreceptor association: role in hepatitis C virus entry. J Virol, 82 (10), pp. 5007-5020. | Show Abstract | Read more

Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.

Jamshad M, Rajesh S, Stamataki Z, McKeating JA, Dafforn T, Overduin M, Bill RM. 2008. Structural characterization of recombinant human CD81 produced in Pichia pastoris. Protein Expr Purif, 57 (2), pp. 206-216. | Show Abstract | Read more

Human CD81 (hCD81) protein has been recombinantly produced in the methylotrophic yeast Pichia pastoris. The purified protein, produced at a yield of 1.75 mg/L of culture, was shown to interact with Hepatitis C virus E2 glycoprotein. Immunofluorescent and flow cytometric staining of P. pastoris protoplasts with monoclonal antibodies specific for the second extracellular loop (EC2) of hCD81 confirmed the antigenicity of the recombinant molecule. Full-length hCD81 was solubilized with an array of detergents and subsequently characterized using circular dichroism (CD) and analytical ultracentrifugation. These biophysical techniques confirmed that the protein solution comprises a homogenous species possessing a highly-defined alpha-helical secondary structure. The predicted alpha-helical content of the protein from CD analysis (77.1%) fits remarkably well with what would be expected (75.2%) from knowledge of the protein sequence together with the data from the crystal structure of the second extracellular loop. This study represents the first biophysical characterization of a full-length recombinant tetraspanin, and opens the way for structure-activity analyses of this ubiquitous family of transmembrane proteins.

Reynolds GM, Harris HJ, Jennings A, Hu K, Grove J, Lalor PF, Adams DH, Balfe P, Hübscher SG, McKeating JA. 2008. Hepatitis C virus receptor expression in normal and diseased liver tissue. Hepatology, 47 (2), pp. 418-427. | Show Abstract | Read more

UNLABELLED: The principal site of hepatitis C virus (HCV) replication is the liver. HCV pseudoparticles infect human liver derived cell lines and this suggests that liver-specific receptors contribute to defining HCV hepatotropism. At least three host cell molecules have been reported to be important for HCV entry: the tetraspanin CD81, scavenger receptor class B member I (SR-BI), and the tight junction (TJ) protein Claudin 1 (CLDN1). Hepatocytes in liver tissue coexpress CD81, SR-BI, and CLDN1, consistent with their ability to support HCV entry. CLDN1 localized at the apical-canalicular TJ region and at basolateral-sinusoidal hepatocyte surfaces in normal tissue and colocalized with CD81 at both sites. In contrast, CLDN1 appeared to colocalize with SR-BI at the basolateral-sinusoidal surface. CLDN1 expression was increased on basolateral hepatocyte membranes in HCV-infected and other chronically inflamed liver tissue compared with normal liver. In contrast, CLDN4 hepatocellular staining was comparable in normal and diseased liver tissue. CONCLUSION: HCV infection of Huh-7.5 hepatoma cells in vitro significantly increased CLDN1 expression levels, consistent with a direct modulation of CLDN1 by virus infection. In HCV infected livers, immunohistochemical studies revealed focal patterns of CLDN1 staining, suggesting localized areas of increased CLDN1 expression in vivo which may potentiate local viral spread within the liver.

Molina S, Castet V, Pichard-Garcia L, Wychowski C, Meurs E, Pascussi J-M, Sureau C, Fabre J-M, Sacunha A, Larrey D et al. 2008. Serum-derived hepatitis C virus infection of primary human hepatocytes is tetraspanin CD81 dependent. J Virol, 82 (1), pp. 569-574. | Show Abstract | Read more

Hepatitis C virus-positive serum (HCVser, genotypes 1a to 3a) or HCV cell culture (JFH1/HCVcc) infection of primary normal human hepatocytes was assessed by measuring intracellular HCV RNA strands. Anti-CD81 antibodies and siRNA-CD81 silencing markedly inhibited (>90%) HCVser infection irrespective of HCV genotype, viral load, or liver donor, while hCD81-large intracellular loop (LEL) had no effect. However, JFH1/HCVcc infection of hepatocytes was modestly inhibited (40 to 60%) by both hCD81-LEL and anti-CD81 antibodies. In conclusion, CD81 is involved in HCVser infection of human hepatocytes, and comparative studies of HCVser versus JFH1/HCVcc infection of human hepatocytes and Huh-7.5 cells revealed that the cell-virion combination is determinant of the entry process.

Law M, Maruyama T, Lewis J, Giang E, Tarr AW, Stamataki Z, Gastaminza P, Chisari FV, Jones IM, Fox RI et al. 2008. Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med, 14 (1), pp. 25-27. | Show Abstract | Read more

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.

Mee CJ, Grove J, Harris HJ, Hu K, Balfe P, McKeating JA. 2008. Effect of cell polarization on hepatitis C virus entry. J Virol, 82 (1), pp. 461-470. | Show Abstract | Read more

The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.

Timpe JM, Stamataki Z, Jennings A, Hu K, Farquhar MJ, Harris HJ, Schwarz A, Desombere I, Roels GL, Balfe P, McKeating JA. 2008. Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies. Hepatology, 47 (1), pp. 17-24. | Show Abstract | Read more

UNLABELLED: Hepatitis C virus (HCV) infection of Huh-7.5 hepatoma cells results in focal areas of infection where transmission is potentiated by cell-cell contact. To define route(s) of transmission, HCV was allowed to infect hepatoma cells in the presence or absence of antibodies that neutralize cell-free virus infectivity. Neutralizing antibodies (nAbs) reduced cell-free virus infectivity by >95% and had minimal effect(s) on the frequency of infected cells in the culture. To assess whether cell-cell transfer of viral infectivity occurs, HCV-infected cells were cocultured with fluorescently labeled naïve cells in the presence or absence of nAbs. Enumeration by flow cytometry demonstrated cell-cell transfer of infectivity in the presence or absence of nAbs and immunoglobulins from HCV(+) patients. The host cell molecule CD81 and the tight junction protein Claudin 1 (CLDN1) are critical factors defining HCV entry. Soluble CD81 and anti-CD81 abrogated cell-free infection of Huh-7.5 and partially inhibited cell-cell transfer of infection. CD81-negative HepG2 hepatoma cells were resistant to cell-free virus infection but became infected after coculturing with JFH-infected cells in the presence of nAb, confirming that CD81-independent routes of cell-cell transmission exist. Further experiments with 293T and 293T-CLDN1 targets suggested that cell-cell transmission is dependent on CLDN1 expression. CONCLUSION: These data suggest that HCV can transmit in vitro by at least two routes, cell-free virus infection and direct transfer between cells, with the latter offering a novel route for evading nAbs.

Stamataki Z, Coates S, Evans MJ, Wininger M, Crawford K, Dong C, Fong Y-L, Chien D, Abrignani S, Balfe P et al. 2007. Hepatitis C virus envelope glycoprotein immunization of rodents elicits cross-reactive neutralizing antibodies. Vaccine, 25 (45), pp. 7773-7784. | Show Abstract | Read more

Neutralizing antibody responses elicited during infection generally confer protection from infection. Hepatitis C virus (HCV) encodes two glycoproteins E1 and E2 that are essential for virus entry and are the major target for neutralizing antibodies. To assess whether both glycoproteins are required for the generation of a neutralizing antibody response, rodents were immunized with a series of glycoproteins comprising full length and truncated versions. Guinea pigs immunized with HCV-1 genotype 1a E1E2p7, E1E2 or E2 generated high titer anti-glycoprotein antibody responses that neutralized the infectivity of HCVpp and HCVcc expressing gps of the same genotype as the immunizing antigen. Less potent neutralization of viruses bearing the genotype 2 strain J6 gps was observed. In contrast, immunized mice demonstrated reduced anti-gp antibody responses, consistent with their minimal neutralizing activity. Immunization with E2 alone was sufficient to induce a high titer response that neutralized HCV pseudoparticles (HCVpp) bearing diverse glycoproteins and cell culture grown HCV (HCVcc). The neutralization titer was reduced 3-fold by the presence of lipoproteins in human sera. Cross-competition of the guinea pig anti-E1E2 immune sera with a panel of epitope mapped anti-E2 monoclonal antibodies for binding E2 identified a series of epitopes within the N-terminal domain that may be immunogenic in the immunized rodents. These data demonstrate that recombinant E2 and E1E2 can induce polyclonal antibody responses with cross-reactive neutralizing activity, supporting the future development of prophylactic and therapeutic vaccines.

Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wölk B, Hatziioannou T, McKeating JA, Bieniasz PD, Rice CM. 2007. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature, 446 (7137), pp. 801-805. | Show Abstract | Read more

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. A better understanding of the viral life cycle, including the mechanisms of entry into host cells, is needed to identify novel therapeutic targets. Although HCV entry requires the CD81 co-receptor, and other host molecules have been implicated, at least one factor critical to this process remains unknown (reviewed in refs 1-3). Using an iterative expression cloning approach we identified claudin-1 (CLDN1), a tight junction component that is highly expressed in the liver, as essential for HCV entry. CLDN1 is required for HCV infection of human hepatoma cell lines and is the first factor to confer susceptibility to HCV when ectopically expressed in non-hepatic cells. Discrete residues within the first extracellular loop (EL1) of CLDN1, but not protein interaction motifs in intracellular domains, are critical for HCV entry. Moreover, antibodies directed against an epitope inserted in the CLDN1 EL1 block HCV infection. The kinetics of this inhibition indicate that CLDN1 acts late in the entry process, after virus binding and interaction with the HCV co-receptor CD81. With CLDN1 we have identified a novel key factor for HCV entry and a new target for antiviral drug development.

Tscherne DM, Evans MJ, von Hahn T, Jones CT, Stamataki Z, McKeating JA, Lindenbach BD, Rice CM. 2007. Superinfection exclusion in cells infected with hepatitis C virus. J Virol, 81 (8), pp. 3693-3703. | Show Abstract | Read more

Superinfection exclusion is the ability of an established virus infection to interfere with infection by a second virus. In this study, we found that Huh-7.5 cells acutely infected with hepatitis C virus (HCV) genotype 2a (chimeric strain J6/JFH) and cells harboring HCV genotype 1a, 1b, or 2a full-length or subgenomic replicons were resistant to infection with cell culture-produced HCV (HCVcc). Replicon-containing cells became permissive for HCVcc infection after treatment with an HCV-specific protease inhibitor. With the exception of cells harboring a J6/JFH-FLneo replicon, infected or replicon-containing cells were permissive for HCV pseudoparticle (HCVpp) entry, demonstrating a postentry superinfection block downstream of primary translation. The surprising resistance of J6/JFH-FLneo replicon-containing cells to HCVpp infection suggested a defect in virus entry. This block was due to reduced expression of the HCV coreceptor CD81. Further analyses indicated that J6/JFH may be toxic for cells expressing high levels of CD81, thus selecting for a CD81(low) population. CD81 down regulation was not observed in acutely infected cells, suggesting that this may not be a general mechanism of HCV superinfection exclusion. Thus, HCV establishes superinfection exclusion at a postentry step, and this effect is reversible by treatment of infected cells with antiviral compounds.

Grove J, Huby T, Stamataki Z, Vanwolleghem T, Meuleman P, Farquhar M, Schwarz A, Moreau M, Owen JS, Leroux-Roels G et al. 2007. Scavenger receptor BI and BII expression levels modulate hepatitis C virus infectivity. J Virol, 81 (7), pp. 3162-3169. | Show Abstract | Read more

Hepatitis C virus (HCV) enters cells via a pH- and clathrin-dependent endocytic pathway. Scavenger receptor BI (SR-BI) and CD81 are important entry factors for HCV internalization into target cells. The SR-BI gene gives rise to at least two mRNA splice variants, SR-BI and SR-BII, which differ in their C termini. SR-BI internalization remains poorly understood, but SR-BII is reported to endocytose via a clathrin-dependent pathway, making it an attractive target for HCV internalization. We demonstrate that HCV soluble E2 can interact with human SR-BI and SR-BII. Increased expression of SR-BI and SR-BII in the Huh-7.5 hepatoma cell line enhanced HCV strain J6/JFH and JFH infectivity, suggesting that endogenous levels of these receptors limit infection. Elevated expression of SR-BI, but not SR-BII, increased the rate of J6/JFH infection, which may reflect altered intracellular trafficking of the splice variants. In human plasma, HCV particles have been reported to be complexed with lipoproteins, suggesting an indirect interaction of the virus with SR-BI and other lipoprotein receptors. Plasma from J6/JFH-infected uPA-SCID mice transplanted with human hepatocytes demonstrates an increased infectivity for SR-BI/II-overexpressing Huh-7.5 cells. Plasma-derived J6/JFH infectivity was inhibited by an anti-E2 monoclonal antibody, suggesting that plasma virus interaction with SR-BI was glycoprotein dependent. Finally, anti-SR-BI antibodies inhibited the infectivity of cell culture- and plasma-derived J6/JFH, suggesting a critical role for SR-BI/II in HCV infection.

von Hahn T, McKeating JA. 2007. In vitro veritas? The challenges of studying hepatitis C virus infectivity in a test tube. J Hepatol, 46 (3), pp. 355-358. | Read more

Kaplan DE, Sugimoto K, Newton K, Valiga ME, Ikeda F, Aytaman A, Nunes FA, Lucey MR, Vance BA, Vonderheide RH et al. 2007. Discordant role of CD4 T-cell response relative to neutralizing antibody and CD8 T-cell responses in acute hepatitis C. Gastroenterology, 132 (2), pp. 654-666. | Show Abstract | Read more

BACKGROUND & AIMS: Acute hepatitis C virus (HCV) infection becomes chronic in the majority of patients. Although HCV-specific CD4 T-cell response is associated with HCV clearance, less is known about virus-specific CD8 T-cell or neutralizing antibody (nAb) responses and the role of CD4 help in their induction during acute infection. METHODS: HCV-specific CD4, CD8, and HCV pseudoparticle (HCVpp) nAb responses were monitored in acutely HCV-infected patients to define their relative contributions to viral clearance. RESULTS: Our results show that the outcome of acute hepatitis C is associated with a functional hierarchy in HCV-specific CD4 T-cell response and the scope of virus-specific, total T-cell interferon-gamma response. HCV-specific CD8 T-cell response was readily detectable in acutely HCV-infected patients regardless of virologic outcome or virus-specific CD4 T-cell response. In contrast, HCVpp-specific nAbs were readily detected in patients with chronic evolution and impaired virus-specific CD4 T-cell response but not in patients who cleared infection with robust virus-specific CD4 T-cell response. CONCLUSIONS: The outcome of acute hepatitis C is associated with efficient virus-specific CD4 T-cell response(s) without which HCV-specific CD8 T-cell and heterologous nAb responses may develop but fail to clear viremia. Furthermore, HCV-specific nAb responses may not be induced despite robust virus-specific CD4 T-cell response.

von Hahn T, Yoon JC, Alter H, Rice CM, Rehermann B, Balfe P, McKeating JA. 2007. Hepatitis C virus continuously escapes from neutralizing antibody and T-cell responses during chronic infection in vivo. Gastroenterology, 132 (2), pp. 667-678. | Show Abstract | Read more

BACKGROUND & AIMS: Broadly reactive neutralizing antibodies (nAbs) and multispecific T-cell responses are generated during chronic hepatitis C virus (HCV) infection and yet fail to clear the virus. This study investigated the development of autologous nAb and HCV-glycoprotein-specific T-cell responses and their effects on viral sequence evolution during chronic infection in order to understand the reasons for their lack of effectiveness. METHODS: Numerous E1E2 sequences were amplified and sequenced from serum samples collected over a 26-year period from patient H, a uniquely well-characterized, chronically infected individual. HCV pseudoparticles (HCVpp) expressing the patient-derived glycoproteins were generated and tested for their sensitivity to neutralization by autologous and heterologous serum antibodies. RESULTS: A strain-specific nAb response developed early in infection (8 weeks postinfection), whereas cross-reactive antibodies able to neutralize HCVpp-bearing heterologous glycoproteins developed late in infection (>33 wk postinfection). The humoral response continuously failed to neutralize viruses bearing autologous glycoprotein sequences that were present in the serum at a given time. The amplified glycoprotein sequences displayed high variability, particularly in regions corresponding to defined linear B-cell epitopes. Mutations in defined neutralizing epitopes were associated with a loss of recognition by monoclonal antibodies against these epitopes and with decreased neutralization of corresponding HCVpp. Viral escape from CD4 and CD8 T-cell responses also was shown for several novel epitopes throughout the glycoprotein region. CONCLUSIONS: During chronic infection HCV is subjected to selection pressures from both humoral and cellular immunity, resulting in the continuous generation of escape variants.

Kapadia SB, Barth H, Baumert T, McKeating JA, Chisari FV. 2007. Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I. J Virol, 81 (1), pp. 374-383. | Show Abstract | Read more

In the past several years, a number of cellular proteins have been identified as candidate entry receptors for hepatitis C virus (HCV) by using surrogate models of HCV infection. Among these, the tetraspanin CD81 and scavenger receptor B type I (SR-BI), both of which localize to specialized plasma membrane domains enriched in cholesterol, have been suggested to be key players in HCV entry. In the current study, we used a recently developed in vitro HCV infection system to demonstrate that both CD81 and SR-BI are required for authentic HCV infection in vitro, that they function cooperatively to initiate HCV infection, and that CD81-mediated HCV entry is, in part, dependent on membrane cholesterol.

Flint M, von Hahn T, Zhang J, Farquhar M, Jones CT, Balfe P, Rice CM, McKeating JA. 2006. Diverse CD81 proteins support hepatitis C virus infection. J Virol, 80 (22), pp. 11331-11342. | Show Abstract | Read more

Hepatitis C virus (HCV) entry is dependent on CD81. To investigate whether the CD81 sequence is a determinant of HCV host range, we expressed a panel of diverse CD81 proteins and tested their ability to interact with HCV. CD81 large extracellular loop (LEL) sequences were expressed as recombinant proteins; the human and, to a low level, the African green monkey sequences bound soluble HCV E2 (sE2) and inhibited infection by retrovirus pseudotype particles bearing HCV glycoproteins (HCVpp). In contrast, mouse or rat CD81 proteins failed to bind sE2 or to inhibit HCVpp infection. However, CD81 proteins from all species, when expressed in HepG2 cells, conferred susceptibility to infection by HCVpp and cell culture-grown HCV to various levels, with the rat sequence being the least efficient. Recombinant human CD81 LEL inhibited HCVpp infectivity only if present during the virus-cell incubation, consistent with a role for CD81 after virus attachment. Amino acid changes that abrogate sE2 binding (I182F, N184Y, and F186S, alone or in combination) were introduced into human CD81. All three amino acid changes in human CD81 resulted in a molecule that still supported HCVpp infection, albeit with reduced efficiency. In summary, there is a remarkable plasticity in the range of CD81 sequences that can support HCV entry, suggesting that CD81 polymorphism may contribute to, but alone does not define, the HCV susceptibility of a species. In addition, the capacity to support viral entry is only partially reflected by assays measuring sE2 interaction with recombinant or full-length CD81 proteins.

Zhong J, Gastaminza P, Chung J, Stamataki Z, Isogawa M, Cheng G, McKeating JA, Chisari FV. 2006. Persistent hepatitis C virus infection in vitro: coevolution of virus and host. J Virol, 80 (22), pp. 11082-11093. | Show Abstract | Read more

The virological and cellular consequences of persistent hepatitis C virus (HCV) infection have been elusive due to the absence of the requisite experimental systems. Here, we report the establishment and the characteristics of persistent in vitro infection of human hepatoma-derived cells by a recently described HCV genotype 2a infectious molecular clone. Persistent in vitro infection was characterized by the selection of viral variants that displayed accelerated expansion kinetics, higher peak titers, and increased buoyant densities. Sequencing analysis revealed the selection of a single adaptive mutation in the HCV E2 envelope protein that was largely responsible for the variant phenotype. In parallel, as the virus became more aggressive, cells that were resistant to infection emerged, displaying escape mechanisms operative at the level of viral entry, HCV RNA replication, or both. Collectively, these results reveal the existence of coevolutionary events during persistent HCV infection that favor survival of both virus and host.

Lai WK, Sun PJ, Zhang J, Jennings A, Lalor PF, Hubscher S, McKeating JA, Adams DH. 2006. Expression of DC-SIGN and DC-SIGNR on human sinusoidal endothelium: a role for capturing hepatitis C virus particles. Am J Pathol, 169 (1), pp. 200-208. | Show Abstract | Read more

Hepatic sinusoidal endothelial cells are unique among endothelial cells in their ability to internalize and process a diverse range of antigens. DC-SIGNR, a type 2 C-type lectin expressed on liver sinusoids, has been shown to bind with high affinity to hepatitis C virus (HCV) E2 glycoprotein. DC-SIGN is a closely related homologue reported to be expressed only on dendritic cells and a subset of macrophages and has similar binding affinity to HCV E2 glycoprotein. These receptors function as adhesion and antigen presentation molecules. We report distinct patterns of DC-SIGNR and DC-SIGN expression in human liver tissue and show for the first time that both C-type lectins are expressed on sinusoidal endothelial cells. We confirmed that these receptors are functional by demonstrating their ability to bind HCV E2 glycoproteins. Although these lectins on primary sinusoidal cells support HCV E2 binding, they are unable to support HCV entry. These data support a model where DC-SIGN and DC-SIGNR on sinusoidal endothelium provide a mechanism for high affinity binding of circulating HCV within the liver sinusoids allowing subsequent transfer of the virus to underlying hepatocytes, in a manner analogous to DC-SIGN presentation of human immunodeficiency virus on dendritic cells.

von Hahn T, Lindenbach BD, Boullier A, Quehenberger O, Paulson M, Rice CM, McKeating JA. 2006. Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells. Hepatology, 43 (5), pp. 932-942. | Show Abstract | Read more

Cell entry of hepatitis C virus, pseudoparticles (HCVpp) and cell culture grown virus (HCVcc), requires the interaction of viral glycoproteins with CD81 and other as yet unknown cellular factors. One of these is likely to be the scavenger receptor class B type I (SR-BI). To further understand the role of SR-BI, we examined the effect of SR-BI ligands on HCVpp and HCVcc infectivity. Oxidized low-density lipoprotein (oxLDL), but not native LDL, potently inhibited HCVpp and HCVcc cell entry. Pseudoparticles bearing unrelated viral glycoproteins or bovine viral diarrhea virus were not affected. A dose-dependent inhibition was observed for HCVpp bearing diverse viral glycoproteins with an approximate IC50 of 1.5 microg/mL apolipoprotein content, which is within the range of oxLDL reported to be present in human plasma. The ability of lipoprotein components to bind to target cells associated with their antiviral activity, suggesting a mechanism of action which targets a cell surface receptor critical for HCV infection of the host cell. However, binding of soluble E2 to SR-BI or CD81 was not affected by oxLDL, suggesting that oxLDL does not act as a simple receptor blocker. At the same time, oxLDL incubation altered the biophysical properties of HCVpp, suggesting a ternary interaction of oxLDL with both virus and target cells. In conclusion, the SR-BI ligand oxLDL is a potent cell entry inhibitor for a broad range of HCV strains in vitro. These findings suggest that SR-BI is an essential component of the cellular HCV receptor complex.

Lindenbach BD, Meuleman P, Ploss A, Vanwolleghem T, Syder AJ, McKeating JA, Lanford RE, Feinstone SM, Major ME, Leroux-Roels G, Rice CM. 2006. Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc Natl Acad Sci U S A, 103 (10), pp. 3805-3809. | Show Abstract | Read more

Hepatitis C virus (HCV) is a major cause of chronic liver disease, frequently progressing to cirrhosis and increased risk of hepatocellular carcinoma. Current therapies are inadequate and progress in the field has been hampered by the lack of efficient HCV culture systems. By using a recently described HCV genotype 2a infectious clone that replicates and produces infectious virus in cell culture (HCVcc), we report here that HCVcc strain FL-J6/JFH can establish long-term infections in chimpanzees and in mice containing human liver grafts. Importantly, virus recovered from these animals was highly infectious in cell culture, demonstrating efficient ex vivo culture of HCV. The improved infectivity of animal-derived HCV correlated with virions of a lower average buoyant density than HCVcc, suggesting that physical association with low-density factors influences viral infectivity. These results greatly extend the utility of the HCVcc genetic system to allow the complete in vitro and in vivo dissection of the HCV life cycle.

Tscherne DM, Jones CT, Evans MJ, Lindenbach BD, McKeating JA, Rice CM. 2006. Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J Virol, 80 (4), pp. 1734-1741. | Show Abstract | Read more

Hepatitis C virus (HCV) is an important human pathogen associated with chronic liver disease. Recently, based on a genotype 2a isolate, tissue culture systems supporting complete replication and infectious virus production have been developed. In this study, we used cell culture-produced infectious HCV to analyze the viral entry pathway into Huh-7.5 cells. Bafilomycin A1 and concanamycin A, inhibitors of vacuolar ATPases, prevented HCV entry when they were present prior to infection and had minimal effect on downstream replication events. HCV entry therefore appears to be pH dependent, requiring an acidified intracellular compartment. For many other enveloped viruses, acidic pH triggers an irreversible conformational change, which promotes virion-endosomal membrane fusion. Such viruses are often inactivated by low pH. In the case of HCV, exposure of virions to acidic pH followed by return to neutral pH did not affect their infectivity. This parallels the observation made for the related pestivirus bovine viral diarrhea virus. Low pH could activate the entry of cell surface-bound HCV but only after prolonged incubation at 37 degrees C. This suggests that there are rate-limiting, postbinding events that are needed to render HCV competent for low-pH-triggered entry. Such events may involve interaction with a cellular coreceptor or other factors but do not require cathepsins B and L, late endosomal proteases that activate Ebola virus and reovirus for entry.

Lindenbach BD, Evans MJ, Syder AJ, Wölk B, Tellinghuisen TL, Liu CC, Maruyama T, Hynes RO, Burton DR, McKeating JA, Rice CM. 2005. Complete replication of hepatitis C virus in cell culture. Science, 309 (5734), pp. 623-626. | Show Abstract | Read more

Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10(5) infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-alpha and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.

McKeating JA, Zhang LQ, Logvinoff C, Flint M, Zhang J, Yu J, Butera D, Ho DD, Dustin LB, Rice CM, Balfe P. 2004. Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner. J Virol, 78 (16), pp. 8496-8505. | Show Abstract | Read more

We recently reported that retroviral pseudotypes bearing the hepatitis C virus (HCV) strain H and Con1 glycoproteins, genotype 1a and 1b, respectively, require CD81 as a coreceptor for virus-cell entry and infection. Soluble truncated E2 cloned from a number of diverse HCV genotypes fail to interact with CD81, suggesting that viruses of diverse origin may utilize different receptors and display altered cell tropism. We have used the pseudotyping system to study the tropism of viruses bearing diverse HCV glycoproteins. Viruses bearing these glycoproteins showed a 150-fold range in infectivity for hepatoma cells and failed to infect lymphoid cells. The level of glycoprotein incorporation into particles varied considerably between strains, generally reflecting the E2 expression level within transfected cells. However, differences in glycoprotein incorporation were not associated with virus infectivity, suggesting that infectivity is not limited by the absolute level of glycoprotein. All HCV pseudotypes failed to infect HepG2 cells and yet infected the same cells after transduction to express human CD81, confirming the critical role of CD81 in HCV infection. Interestingly, these HCV pseudotypes differed in their ability to infect HepG2 cells expressing a panel of CD81 variants, suggesting subtle differences in the interaction of CD81 residues with diverse viral glycoproteins. Our current model of HCV infection suggests that CD81, together with additional unknown liver specific receptor(s), mediate the virus-cell entry process.

Logvinoff C, Major ME, Oldach D, Heyward S, Talal A, Balfe P, Feinstone SM, Alter H, Rice CM, McKeating JA. 2004. Neutralizing antibody response during acute and chronic hepatitis C virus infection. Proc Natl Acad Sci U S A, 101 (27), pp. 10149-10154. | Show Abstract | Read more

Little is known about the role of Abs in determining the outcome of hepatitis C virus (HCV) infection. By using infectious retroviral pseudotypes bearing HCV glycoproteins, we measured neutralizing Ab (nAb) responses during acute and chronic HCV infection. In seven acutely infected health care workers, only two developed a nAb response that failed to associate with viral clearance. In contrast, the majority of chronically infected patients had nAbs. To determine the kinetics of strain-specific and crossreactive nAb emergence, we studied patient H, the source of the prototype genotype 1a H77 HCV strain. An early weak nAb response, specific for the autologous virus, was detected at seroconversion. However, neutralization of heterologous viruses was detected only between 33 and 111 weeks of infection. We also examined the development of nAbs in 10 chimpanzees infected with H77 clonal virus. No nAb responses were detected in three animals that cleared virus, whereas strain-specific nAbs were detected in six of the seven chronically infected animals after approximately 50 weeks of infection. The delayed appearance of high titer crossreactive nAbs in chronically infected patients suggests that selective mechanism(s) may operate to prevent the appearance of these Abs during acute infection. The long-term persistence of these nAbs in chronically infected patients may regulate viral replication.

Flint M, Logvinoff C, Rice CM, McKeating JA. 2004. Characterization of infectious retroviral pseudotype particles bearing hepatitis C virus glycoproteins. J Virol, 78 (13), pp. 6875-6882. | Show Abstract | Read more

The recent development of infectious retroviral pseudotypes bearing hepatitis C virus (HCV) glycoproteins represents an opportunity to study the functionally active form of the HCV E1 and E2 glycoproteins. In the culture supernatant of cells producing HCV retroviral pseudotypes, the majority of E2 was not associated with infectious particles and failed to sediment on sucrose gradients. The E2 that was incorporated into infectious particles appeared as a triplet of diffuse bands at 60, 70, and 90 kDa. Similarly, three major forms of E1 were incorporated into the pseudotype particles, migrating at 33, 31, and 25 kDa. Endoglycosidase H (endo-H) treatment of particles demonstrated that the incorporated E1 was partially or completely sensitive to digestion. In contrast, the majority of the incorporated E2 was endo-H resistant. Purified pseudotype particles were found to contain both disulfide-linked aggregates and nonaggregated E1 and E2. HCV pseudotypes generated from cells expressing E1E2p7 showed similar heterogeneity in the incorporated glycoproteins and were of comparable infectivity to those generated by expression of E1E2. Our results demonstrate the heterogeneous nature of E1 and E2 incorporated into retroviral pseudotypes and highlight the difficulty in identifying forms of the HCV glycoproteins that initiate infection.

Zhang J, Randall G, Higginbottom A, Monk P, Rice CM, McKeating JA. 2004. CD81 is required for hepatitis C virus glycoprotein-mediated viral infection. J Virol, 78 (3), pp. 1448-1455. | Show Abstract | Read more

CD81 has been described as a putative receptor for hepatitis C virus (HCV); however, its role in HCV cell entry has not been characterized due to the lack of an efficient cell culture system. We have examined the role of CD81 in HCV glycoprotein-dependent entry by using a recently developed retroviral pseudotyping system. Human immunodeficiency virus (HIV) pseudotypes bearing HCV E1E2 glycoproteins show a restricted tropism for human liver cell lines. Although all of the permissive cell lines express CD81, CD81 expression alone is not sufficient to allow viral entry. CD81 is required for HIV-HCV pseudotype infection since (i) a monoclonal antibody specific for CD81 inhibited infection of susceptible target cells and (ii) silencing of CD81 expression in Huh-7.5 hepatoma cells by small interfering RNAs inhibited HIV-HCV pseudotype infection. Furthermore, expression of CD81 in human liver cells that were previously resistant to infection, HepG2 and HH29, conferred permissivity of HCV pseudotype infection. The characterization of chimeric CD9/CD81 molecules confirmed that the large extracellular loop of CD81 is a determinant for viral entry. These data suggest a functional role for CD81 as a coreceptor for HCV glycoprotein-dependent viral cell entry.

Hsu M, Zhang J, Flint M, Logvinoff C, Cheng-Mayer C, Rice CM, McKeating JA. 2003. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci U S A, 100 (12), pp. 7271-7276. | Show Abstract | Read more

HIV pseudotypes bearing native hepatitis C virus (HCV) glycoproteins (strain H and Con1) are infectious for the human hepatoma cell lines Huh-7 and PLC/PR5. Infectivity depends on coexpression of both E1 and E2 glycoproteins, is pH-dependent, and can be neutralized by mAbs mapping to amino acids 412-447 within E2. Cell-surface expression of one or all of the candidate receptor molecules (CD81, low-density lipoprotein receptor, scavenger receptor class B type 1, and dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin) failed to confer permissivity to HIV-HCV pseudotype infection. However, HIV-HCV pseudotype infectivity was inhibited by a recombinant soluble form of CD81 and a mAb specific for CD81, suggesting that CD81 may be a component of a receptor complex.

Pöhlmann S, Zhang J, Baribaud F, Chen Z, Leslie GJ, Lin G, Granelli-Piperno A, Doms RW, Rice CM, McKeating JA. 2003. Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR. J Virol, 77 (7), pp. 4070-4080. | Show Abstract | Read more

DC-SIGN and DC-SIGNR are two closely related membrane-associated C-type lectins that bind human immunodeficiency virus (HIV) envelope glycoprotein with high affinity. Binding of HIV to cells expressing DC-SIGN or DC-SIGNR can enhance the efficiency of infection of cells coexpressing the specific HIV receptors. DC-SIGN is expressed on some dendritic cells, while DC-SIGNR is localized to certain endothelial cell populations, including hepatic sinusoidal endothelial cells. We found that soluble versions of the hepatitis C virus (HCV) E2 glycoprotein and retrovirus pseudotypes expressing chimeric forms of both HCV E1 and E2 glycoproteins bound efficiently to DC-SIGN and DC-SIGNR expressed on cell lines and primary human endothelial cells but not to other C-type lectins tested. Soluble E2 bound to immature and mature human monocyte-derived dendritic cells (MDDCs). Binding of E2 to immature MDDCs was dependent on DC-SIGN interactions, while binding to mature MDDCs was partly independent of DC-SIGN, suggesting that other cell surface molecules may mediate HCV glycoprotein interactions. HCV interactions with DC-SIGN and DC-SIGNR may contribute to the establishment or persistence of infection both by the capture and delivery of virus to the liver and by modulating dendritic cell function.

Blight KJ, McKeating JA, Marcotrigiano J, Rice CM. 2003. Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. J Virol, 77 (5), pp. 3181-3190. | Show Abstract | Read more

Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.

Blight KJ, McKeating JA, Rice CM. 2002. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol, 76 (24), pp. 13001-13014. | Show Abstract | Read more

Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-alpha) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.

Jeffs SA, Shotton C, Balfe P, McKeating JA. 2002. Truncated gp120 envelope glycoprotein of human immunodeficiency virus 1 elicits a broadly reactive neutralizing immune response. J Gen Virol, 83 (Pt 11), pp. 2723-2732. | Show Abstract | Read more

Removal of the V1-V3 loops from IIIB gp120 results in a protein, PR12, with altered immunogenicity compared to the full-length protein. Polyclonal immune sera raised in rats using PR12 as immunogen recognizes envelope glycoproteins of clades A, B, C, E, F and G and can neutralize chimeric human immunodeficiency virus type 1 (HIV-1) HXB2 viruses expressing envelopes from primary HIV-1 clades B, C, E and F. These data suggest that the immune response to PR12 is directed toward conserved epitopes expressed by viral glycoproteins of diverse genotypes. Five monoclonal antibodies (mAb) derived from PR12-immunized rats were unable to neutralize virus infectivity; hence the epitopes responsible for the induction of this cross-clade neutralizing activity remain to be elucidated. However, PR12 immune sera were able to compete with the human neutralizing mAb 2G12 for gp120 binding, implying that this epitope may be immunogenic when expressed in the context of this truncated protein.

Mueller A, Mahmoud NG, Goedecke MC, McKeating JA, Strange PG. 2002. Pharmacological characterization of the chemokine receptor, CCR5. Br J Pharmacol, 135 (4), pp. 1033-1043. | Show Abstract | Read more

1. We investigated the effects of a number of naturally occurring chemokines (MIP-1alpha, MIP-1beta, RANTES, MCP-2, MCP-3, MCP-4) on different processes linked to the chemokine receptor CCR5 in recombinant CHO cells expressing the receptor at different levels. 2. Internalization of CCR5 following chemokine treatment was studied and MIP-1alpha, MIP-1beta and RANTES (50 nM) were able to induce internalization (similar50%) of the receptor. Internalization due to MCP-2, MCP-3 and MCP-4 was less (similar20%). 3. Phosphorylation of CCR5 following chemokine treatment was studied and MIP-1alpha, MIP-1beta and RANTES (50 nM) were able to induce phosphorylation of CCR5 whereas the other chemokines did not induce CCR5 phosphorylation. 4. MIP-1alpha, MIP-1beta, RANTES and MCP-2 were able to stimulate [(35)S]-GTPgammaS binding, an index of receptor/G protein activation, whereas MCP-3 and MCP-4 had no effect in this assay. MCP-2 was a partial agonist (similar80%) compared to MIP-1alpha, MIP-1beta and RANTES, which gave similar maximal stimulations in this assay. 5. MIP-1alpha, MIP-1beta, RANTES, MCP-2 and MCP-4 were able to stimulate increases in intracellular calcium ions via activation of CCR5 whereas MCP-3 was without effect. 6. It is concluded that different chemokines interacting with CCR5 mediate different patterns of cellular responses.

Owsianka A, Clayton RF, Loomis-Price LD, McKeating JA, Patel AH. 2001. Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2. J Gen Virol, 82 (Pt 8), pp. 1877-1883. | Show Abstract | Read more

Structure-function analysis of the hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, has been difficult due to the unavailability of HCV virions. Truncated soluble forms of E2 have been used as models to study virus interaction with the putative HCV receptor CD81, but they may not fully mimic E2 structures on the virion. Here, we compared the CD81-binding characteristics of truncated E2 (E2(660)) and full-length (FL) E1E2 complex expressed in mammalian cells, and of HCV virus-like particles (VLPs) generated in insect cells. All three glycoprotein forms interacted with human CD81 in an in vitro binding assay, allowing us to test a panel of well-characterized anti-E2 monoclonal antibodies (MAbs) for their ability to inhibit the glycoprotein-CD81 interaction. MAbs specific for E2 amino acid (aa) regions 396-407, 412-423 and 528-535 blocked binding to CD81 of all antigens tested. However, MAbs specific for regions 432-443, 436-443 and 436-447 inhibited the interaction of VLPs, but not of E2(660) or the FL E1E2 complex with CD81, indicating the existence of structural differences amongst the E2 forms. These findings underscore the need to carefully select an appropriate ligand for structure-function analysis.

Hammond AL, Lewis J, May J, Albert J, Balfe P, McKeating JA. 2001. Antigenic variation within the CD4 binding site of human immunodeficiency virus type 1 gp120: effects on chemokine receptor utilization. J Virol, 75 (12), pp. 5593-5603. | Show Abstract | Read more

To assess the antigenicity of envelope glycoproteins derived from primary human immunodeficiency virus type 1 populations, their interactions with the receptor CD4, and their coreceptor usage, we have cloned and expressed multiple gp120 proteins from a number of primary virus isolates. Characterization of these proteins showed a high degree of antigenic polymorphism both within the CD4 binding site and in defined neutralization epitopes, which may partially account for the general resistance of primary isolates to neutralizing agents. Furthermore, chimeric viruses expressing gp120 proteins with reduced CD4 binding abilities are viable, suggesting that primary viruses may require a less avid interaction with the receptor CD4 to initiate infection than do their laboratory-adapted counterparts. The coreceptor usage of chimeric viruses was related to the ability of the virus to bind CD4, with reduced CD4 binding correlating with preferential usage of CXCR4. Changes in coreceptor usage mapped to sequence changes in the C2 and V4 regions, with no changes seen in the V3 region.

Patel AH, Wood J, Penin F, Dubuisson J, McKeating JA. 2000. Construction and characterization of chimeric hepatitis C virus E2 glycoproteins: analysis of regions critical for glycoprotein aggregation and CD81 binding. J Gen Virol, 81 (Pt 12), pp. 2873-2883. | Show Abstract | Read more

We compared the ability of two closely related truncated E2 glycoproteins (E2(660)) derived from hepatitis C virus (HCV) genotype 1a strains Glasgow (Gla) and H77c to bind a panel of conformation-dependent monoclonal antibodies (MAbs) and CD81. In contrast to H77c, Gla E2(660) formed disulfide-linked high molecular mass aggregates and failed to react with conformation-dependent MAbs and CD81. To delineate amino acid (aa) regions associated with protein aggregation and CD81 binding, several Gla-H77c E2(660) chimeric glycoproteins were constructed. Chimeras C1, C2 and C6, carrying aa 525-660 of Gla E2(660), produced disulfide-linked aggregates and failed to bind CD81 and conformation-dependent MAbs, suggesting that amino acids within this region are responsible for protein misfolding. The presence of Gla hypervariable region 1 (aa 384-406) on H77 E2(660), chimera C4, had no effect on protein folding or CD81 binding. Chimeras C3 and C5, carrying aa 384-524 or 407-524 of Gla E2(660), respectively, were recognized by conformation-dependent MAbs and yet failed to bind CD81, suggesting that amino acids in region 407-524 are important in modulating CD81 interaction without affecting antigen folding. Comparison of Gla and H77c E2(660) aa sequences with those of genotype 1a and divergent genotypes identified a number of variant amino acids, including two putative N-linked glycosylation sites at positions 476 and 532. However, introduction of G476N-G478S and/or D532N in Gla E2(660) had no effect on antigenicity or aggregation.

Chan-Fook C, Jiang WR, Clarke BE, Zitzmann N, Maidens C, McKeating JA, Jones IM. 2000. Hepatitis C virus glycoprotein E2 binding to CD81: the role of E1E2 cleavage and protein glycosylation in bioactivity. Virology, 273 (1), pp. 60-66. | Show Abstract | Read more

The hepatitis C virus glycoproteins E1 and 2 have been expressed using recombinant baculoviruses following fusion to the carrier protein glutathione S-transferase (GST). Proteins were expressed singly and as an E1E2 polyprotein with and without an N-terminal affinity tag. Expression of the E1E2 polyprotein, even when preceded by GST, led to processing in insect cells and detection of an E1E2 complex that could be specifically purified by glutathione affinity chromatography. Baculovirus expressed E2 and a purified GST-E1E2 protein bound to the second extracellular loop of CD81 (EC2), a reported ligand for the molecule, but not to a truncated derivative of CD81 consisting of only the central domain of the loop. Purified GST-E2, however, failed to bind to CD81 suggesting a requirement for a free E2 amino terminus for biological activity. The binding to CD81 by baculovirus expressed E2 protein was comparable to that observed for E2 derived from mammalian cells when detected by a monoclonal antibody sensitive to protein conformation. Furthermore, E2 protein expressed in insect cells in the presence of N-butyldeoxynojirimycin, an inhibitor of terminal glucose residue processing, formed complexes with E1 and bound to CD81-EC2 similarly to untreated protein. Together these data suggest that although hyperglucosylation of E2 does not have a major effect on bioactivity, polyprotein processing to reveal the free amino terminus is required.

Flint M, McKeating JA. 2000. The role of the hepatitis C virus glycoproteins in infection. Rev Med Virol, 10 (2), pp. 101-117. | Show Abstract | Read more

HCV encodes two glycoproteins, E1 and E2, that are believed to be exposed on the surface of virions. These molecules are likely to be involved in viral interactions with the host immune response and responsible for mediating viral entry into target cells. They are obvious major components for prototype vaccine studies. Recently, E2 has been reported to bind to the tetraspan molecule CD81, which represents a putative receptor for HCV. Here, we discuss the role the HCV gps may play during infection, the contribution of E2 gp variation to HCV evasion from the immune response and possible implications of the E2-CD81 interaction for HCV pathogenesis.

Higginbottom A, Quinn ER, Kuo CC, Flint M, Wilson LH, Bianchi E, Nicosia A, Monk PN, McKeating JA, Levy S. 2000. Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2. J Virol, 74 (8), pp. 3642-3649. | Show Abstract | Read more

Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).

Flint M, Dubuisson J, Maidens C, Harrop R, Guile GR, Borrow P, McKeating JA. 2000. Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein. J Virol, 74 (2), pp. 702-709. | Show Abstract | Read more

The E2 protein of hepatitis C virus (HCV) is believed to be a virion surface glycoprotein that is a candidate for inclusion in an antiviral vaccine. A truncated soluble version of E2 has recently been shown to interact with CD81, suggesting that this protein may be a component of the receptor for HCV. When expressed in eukaryotic cells, a significant proportion of E2 forms misfolded aggregates. To analyze the specificity of interaction between E2 and CD81, the aggregated and monomeric forms of a truncated E2 glycoprotein (E2(661)) were separated by high-pressure liquid chromatography and analyzed for CD81 binding. Nonaggregated forms of E2 preferentially bound CD81 and a number of conformation-dependent monoclonal antibodies (MAbs). Furthermore, intracellular forms of E2(661) were found to bind CD81 with greater affinity than the extracellular forms. Intracellular and secreted forms of E2(661) were also found to differ in reactivity with MAbs and human sera, consistent with differences in antigenicity. Together, these data indicate that proper folding of E2 is important for its interaction with CD81 and that modifications of glycans can modulate this interaction. Identification of the biologically active forms of E2 will assist in the future design of vaccines to protect against HCV infection.

Flint M, Maidens C, Loomis-Price LD, Shotton C, Dubuisson J, Monk P, Higginbottom A, Levy S, McKeating JA. 1999. Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81. J Virol, 73 (8), pp. 6235-6244. | Show Abstract

A truncated soluble form of the hepatitis C virus E2 glycoprotein, E2661, binds specifically to the surface of cells expressing human CD81 (hCD81) but not other members of the tetraspanin family (CD9, CD63, and CD151). No differences were noted between the level of E2661 binding to hCD81 expressed on the surface of rat RBL or KM3 cells compared to Daudi and Molt-4 cells, suggesting that additional human-cell-specific factors are not required for the primary interaction of E2 with the cell surface. E2 did not interact with African green monkey (AGM) CD81 on the surface of COS cells, which differs from the hCD81 sequence at four residues within the second extracellular region (EC2) (amino acids [aa] 163, 186, 188, and 196), suggesting that one or more of these residues defines the site of interaction with E2. Various recombinant forms of CD81 EC2 show differences in the ability to bind E2, suggesting that CD81 conformation is important for E2 recognition. Regions of E2 involved in the CD81 interaction were analyzed, and our data suggest that the binding site is of a conformational nature involving aa 480 to 493 and 544 to 551 within the E2 glycoprotein. Finally, we demonstrate that ligation of CD81 by E2661 induced aggregation of lymphoid cells and inhibited B-cell proliferation, demonstrating that E2 interaction with CD81 can modulate cell function.

Flint M, McKeating JA. 1999. The C-terminal region of the hepatitis C virus E1 glycoprotein confers localization within the endoplasmic reticulum. J Gen Virol, 80 ( Pt 8) (8), pp. 1943-1947. | Show Abstract | Read more

Expression of the hepatitis C virus glycoprotein E1 in cultured cells localizes it to the endoplasmic reticulum, suggesting that E1 contains a signal mediating retention. Fusion of the C-terminal region of E1 to the ectodomain of CD4 prevented it from being transported to the cell surface. Fusion of this region of E1 resulted in localization of CD4 and influenza virus haemagglutinin chimeric molecules to a pre-medial Golgi compartment. This signal was present within E1 residues 311-383. Retention was not due to misfolding since the chimeric molecules did not form disulphide-linked aggregates indicative of misfolded proteins, and could be recognized by MAbs specific for conformational epitopes.

Flint M, Thomas JM, Maidens CM, Shotton C, Levy S, Barclay WS, McKeating JA. 1999. Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. J Virol, 73 (8), pp. 6782-6790. | Show Abstract

Hepatitis C virus (HCV) glycoproteins E1 and E2, when expressed in eukaryotic cells, are retained in the endoplasmic reticulum (ER). C-terminal truncation of E2 at residue 661 or 715 (position on the polyprotein) leads to secretion, consistent with deletion of a proposed hydrophobic transmembrane anchor sequence. We demonstrate cell surface expression of a chimeric glycoprotein consisting of E2 residues 384 to 661 fused to the transmembrane and cytoplasmic domains of influenza A virus hemagglutinin (HA), termed E2661-HATMCT. The E2661-HATMCT chimeric glycoprotein was able to bind a number of conformation-dependent monoclonal antibodies and a recombinant soluble form of CD81, suggesting that it was folded in a manner comparable to "native" E2. Furthermore, cell surface-expressed E2661-HATMCT demonstrated pH-dependent changes in antigen conformation, consistent with an acid-mediated fusion mechanism. However, E2661-HATMCT was unable to induce cell fusion of CD81-positive HEK cells after neutral- or low-pH treatment. We propose that a stretch of conserved, hydrophobic amino acids within the E1 glycoprotein, displaying similarities to flavivirus and paramyxovirus fusion peptides, may constitute the HCV fusion peptide. We demonstrate that influenza virus can incorporate E2661-HATMCT into particles and discuss experiments to address the relevance of the E2-CD81 interaction for HCV attachment and entry.

Giorgi JV, Hultin LE, McKeating JA, Johnson TD, Owens B, Jacobson LP, Shih R, Lewis J, Wiley DJ, Phair JP et al. 1999. Shorter survival in advanced human immunodeficiency virus type 1 infection is more closely associated with T lymphocyte activation than with plasma virus burden or virus chemokine coreceptor usage. J Infect Dis, 179 (4), pp. 859-870. | Show Abstract | Read more

To define predictors of survival time in late human immunodeficiency virus type 1 (HIV-1) disease, long- and short-duration survivors were studied after their CD4+ T cells fell to </=50/mm3. Immune activation of CD4+ and CD8+ T cells, as measured by elevated cell surface expression of CD38 antigen, was strongly associated with shorter subsequent survival (P</=.002). The naive CD45RA+CD62L+ T cell reserve was low in all subjects and did not predict survival (P=.34 for CD4+ and.08 for CD8+ cells). Higher virus burden correlated with CD8+ but not CD4+ cell activation and, after correcting for multiple comparisons, was not associated with shorter survival (P=.02). All of the patients' viruses used CCR5, CXCR4, or both, and coreceptor usage did not predict survival (P=. 27). Through mechanisms apparently unrelated to higher virus burden, immune activation is a major determinant of survival in advanced HIV-1 disease.

McKeating JA, Balfe P. 1999. The role of the viral glycoprotein in HIV-1 persistence. Immunol Lett, 65 (1-2), pp. 63-70. | Show Abstract | Read more

The study of the immunological defects which arise from HIV infection has led to a deeper understanding both of the normal immune system and of the mechanisms by which it is damaged in disease. The interactions between viral and host factors during the early stages of HIV infection leads to a post-seroconversion steady state or 'set point' of viral RNA load, which has been shown to be a prognostic marker for subsequent progression rates, further underlining the important role of early immunological responses in the disease process. The changing immune response during the course of infection, together with the changing patterns of antigenicity and tropism leads to a complex series of evolutionary interactions which can be monitored as a series of changes in the properties of the virus over time. Furthermore, significant differences may be seen between the antigenicity of viruses adapted to grow in tissue culture and viruses cultured only briefly in primary cells, and also between the antigenicity of monomeric and oligomeric subunit immunogens. The immunodominant, highly polymorphic and rapidly changing envelope glycoproteins of HIV remains the single biggest target for the design of successful candidate vaccines. The recent crystallisation of one HIV envelope, the proven existence of functionally conserved neutralisation targets and our increasing knowledge of the behaviour of the envelope glycoprotein in vivo offers the possibility that the next generation of vaccine candidates will have a far higher chance of success than has currently been achieved.

Peet NM, McKeating JA, de Souza JB, Roitt IM, Delves PJ, Lund T. 1998. The effect of low-profile serine substitutions in the V3 loop of HIV-1 gp120 IIIB/LAI on the immunogenicity of the envelope protein. Virology, 251 (1), pp. 59-70. | Show Abstract | Read more

Many microbial antigens contain powerful hypervariable epitopes that fail to induce broadly protective immunity because they dominate the immune response at the expense of more conserved but weaker epitopes. If the undesired B cell epitopes are eliminated, the immune system could be focused on the conserved epitopes and produce a stronger antibody response to conserved parts of the protein and thus become a more efficacious immunogen for a vaccine. We examined this possibility using the human immunodeficiency virus envelope glycoprotein (gp)120 IIIB/LAI and selectively replaced the amino acids from the V3 region and analyzed the overall immunogenicity of the mutant proteins after nucleic acid immunization in mice. The most variable residues of the human immunodeficiency virus type 1 gp120 V3 loop sequence were replaced with serine, which has a small uncharged hydrophilic side chain and therefore is likely to be less immunogenic than amino acids found in wildtype V3 sequences. The serine substitutions did not affect the ability of soluble CD4 to bind the mutant molecules compared with wildtype gp120 and monoclonal antibodies against both linear and discontinuous epitopes located in the V1/V2, C1, and C4 regions of the molecule. These data suggest that the V3 loop substitutions did not grossly affect the overall conformation of the envelope molecule. Immunization of CBA x BALB/c F1 mice with DNA expression plasmids for the wild-type gp120 sequence induced a predominantly IgGI antibody response with end point titers of 10(4)-5 x 10(4). The antibodies reacted only with conformationally intact gp120. Serine replacements targeted to both sides of the V3 loop had a major impact on gp120 immunogenicity, with a markedly reduced response in the majority of animals tested. Analysis of the epitope specificity of the responses suggests that N-terminal amino acids in the V3 loop contribute to the major immunodominant epitope and provides no evidence that their removal enhances immunogenicity of the conserved regions.

Lewis J, Balfe P, Arnold C, Kaye S, Tedder RS, McKeating JA. 1998. Development of a neutralizing antibody response during acute primary human immunodeficiency virus type 1 infection and the emergence of antigenic variants. J Virol, 72 (11), pp. 8943-8951. | Show Abstract

We monitored the primary humoral response to human immunodeficiency virus type 1 infection and showed that, in addition to antibodies to p24 and gp41, antigens which form the basis of most diagnostic assays, the response included a significant antibody response directed to the gp120 region of the infecting viral quasispecies. When tested in a recombinant virus neutralization assay, these antibodies were capable of inhibiting viral growth. We found the primary viral quasispecies to solely utilize the CCR-5 chemokine receptor; however, recombinant viruses differed in their cytopathology and in their sensitivity to beta-chemokine inhibition of viral growth. Sequence analysis of the gp120 open reading frames showed that amino acid changes in the C1 (D-->G at position 62) and C4 (V-->A at position 430) regions accounted for the phenotypic differences. These data demonstrate that early in infection, polymorphism exists in envelope glycoprotein coreceptor interactions and imply that therapeutic strategies targeted at this step in the viral life cycle may lead to rapid resistance.

Balfe P, Churcher Y, Penny M, Easterbrook PJ, Goodall RL, Galpin S, Gotch F, Daniels RS, McKeating JA. 1998. Association between a defective CCR-5 gene and progression to disease in HIV infection. AIDS Res Hum Retroviruses, 14 (14), pp. 1229-1234. | Show Abstract | Read more

We measured the effect(s) of CCR-5 genotype on disease progression by studying the frequency of a defective CCR-5 delta32 allele within a cohort of long-term infected individuals. An elevated frequency of CCR-5 delta32 heterozygotes within the cohort compared with a control population of blood donors was observed. An association between progression rate and CCR-5 delta32 heterozygosity was observed. Furthermore, analysis of proviral DNA V3 sequences from a subset of the cohort predicted that the majority of individuals (39 of 44) were infected with viruses predicted to utilize the beta-chemokine receptor CCR-5. The marked association between CCR-5 genotype and disease progression observed in this study may be a consequence of the predicted low frequency of CXCR-4-utilizing viruses present within the selected cohort.

Zhang YJ, Fredriksson R, McKeating JA, Fenyö EM. 1997. Passage of HIV-1 molecular clones into different cell lines confers differential sensitivity to neutralization. Virology, 238 (2), pp. 254-264. | Show Abstract | Read more

In this study, progeny viruses of four HIV-1 molecular clones were tested for sensitivity to neutralization following prolonged passage in peripheral blood mononuclear cells (PBMC) and MT-2, H9, and CEM T-lymphoid cell lines. Two of the viruses were able to establish persistent infection with no cytopathic effect in H9 and CEM cells. Such adaptation conferred increased sensitivity to neutralization by a panel of human sera obtained from HIV-1-infected asymptomatic individuals, by soluble CD4 and by monoclonal antibodies directed to a linear epitope in the V3 region (268-D) and a conformational epitope in the CD4 binding site of the envelope gp 120 (1.5e). Increased sensitivity to neutralization was paralleled by increased binding of these mAbs to native envelope glycoproteins and by increased binding capacity to CD4 expressed on the cell surface. Our results show that virus-host cell interactions are important in influencing sensitivity to neutralization of HIV-1. In primary PBMC or in cytopathic interactions in cell lines, like in MT-2 cells, envelope epitopes important for neutralization remain masked. In contrast, noncytopathic but productive virus-host cell interactions may lead to an increased exposure of neutralizing epitopes and more efficient binding capacity to CD4 resulting in an increased sensitivity to neutralization.

Peet NM, McKeating JA, Ramos B, Klonisch T, De Souza JB, Delves PJ, Lund T. 1997. Comparison of nucleic acid and protein immunization for induction of antibodies specific for HIV-1 gp120. Clin Exp Immunol, 109 (2), pp. 226-232. | Show Abstract | Read more

We have compared the antibody response to HIV-1 gp120 type LAI in mice immunized with either a gp120 expression plasmid or with baculovirus-derived recombinant gp120 (rgp120) formulated with Freund's complete adjuvant. TiterMax, Alum, Ribi R-700, AF-A or QuilA. DNA immunization resulted in variable levels of antibody, with endpoint titres ranging from 10(4) to 10(5), whereas mice immunized with rgp 120 mixed with Ribi R-700, AF-A or QuilA produced antibody levels with endpoint titres > 10(5). Both types of immunization failed to elicit antibodies able to recognize denatured rgp120. The V3 region was immunogenic in animals immunized with nucleic acid, whereas only a few animals immunized with recombinant protein produced antibodies specific for V3 or other linear epitopes, irrespective of the adjuvant used. These data suggest that the immunogenicity of gp120 is dependent upon the mode of antigen delivery, and that in vivo expressed gp120 following nucleic acid immunization elicits, at least with respect to V3, an antibody response which more closely reflects that seen following natural infection in man.

Cooke SJ, Coates K, Barton CH, Biggs TE, Barrett SJ, Cochrane A, Oliver K, McKeating JA, Harris MP, Mann DA. 1997. Regulated expression vectors demonstrate cell-type-specific sensitivity to human immunodeficiency virus type 1 Nef-induced cytostasis. J Gen Virol, 78 ( Pt 2) (2), pp. 381-392. | Show Abstract | Read more

The nef gene product of both human and simian immunodeficiency viruses is critically important for virus replication and disease progression in vivo. However, the precise biological function of Nef remains poorly characterized in vitro, with previous reports suggesting that Nef might be either cytotoxic or cytostatic. As a result of difficulties encountered by several groups in establishing cell lines constitutively expressing Nef, we have developed two inducible systems resulting in stable Nef expression in various mammalian cell lines. Tetracycline-regulated Nef expression was achieved in HeLa cells but could not be established in human T cell lines. Jurkat E6-1 T cell and RAW264.7 murine macrophage cell lines expressing a regulated nef gene were generated using a system in which Nef expression was controlled by a mutated version of the heavy metal-inducible human metallothionein IIA promoter. Induction of high levels of Nef expression in HeLa-Nef and Jurkat-Nef cells resulted in a moderate (2-fold) and a dramatic (10-fold) retardation of cell growth respectively, supporting the contention that Nef may be a cytotoxic or cytostatic factor. This property was also observed at low basal levels of Nef expression in RAW264.7-Nef macrophage clones (5-fold reduction in growth) and was associated with an altered morphological phenotype suggesting that different cell types may be more susceptible to the cytostatic activity of Nef. The regulated Nef-expression systems provide tools for investigating the molecular basis of Nef function, including Nef-mediated cytopathogenicity, CD4 down-regulation and enhancement of virus infectivity.

Fox DG, Balfe P, Palmer CP, May JC, Arnold C, McKeating JA. 1997. Length polymorphism within the second variable region of the human immunodeficiency virus type 1 envelope glycoprotein affects accessibility of the receptor binding site. J Virol, 71 (1), pp. 759-765. | Show Abstract

Sequential mutations were introduced into the V2 region of human immunodeficiency virus (HIV) type 1 HXB2, affecting the length, charge, and number of potential glycosylation sites. The insertions had no effect on cytopathicity or on the ability of virus to replicate in peripheral blood mononuclear cells and established T-cell lines. However, deletion of amino acids 186 to 188, encoding a conserved glycosylation site, resulted in a nonviable virus, suggesting a minimal length requirement of 40 amino acids for a functional V2 loop. However, all amino acid insertions affected the sensitivity of the variants to neutralization by soluble CD4 and monoclonal antibodies specific for epitopes in the V3 and CD4 binding site regions. Furthermore, these mutant viruses showed resistance to neutralization by HIV-positive human sera. Soluble gp120 mutant glycoproteins showed increased affinities for soluble CD4 and monoclonal antibodies specific for a number of epitopes overlapping the CD4 binding site, confirming that length increases in V2 affect exposure of the CD4 binding site. In summary, these data demonstrate that differences in V2 length modulate immunoreactivity of the envelope glycoprotein and support an association between the V2 and CD4 binding site regions.

Israel ZR, Gorny MK, Palmer C, McKeating JA, Zolla-Pazner S. 1997. Prevalence of a V2 epitope in clade B primary isolates and its recognition by sera from HIV-1-infected individuals. AIDS, 11 (1), pp. 128-130.

McKeating JA. 1996. Biological consequences of human immunodeficiency virus type 1 envelope polymorphism: does variation matter? 1995 Fleming Lecture. J Gen Virol, 77 ( Pt 12) (12), pp. 2905-2919. | Read more

McKeating JA, Zhang YJ, Arnold C, Frederiksson R, Fenyö EM, Balfe P. 1996. Chimeric viruses expressing primary envelope glycoproteins of human immunodeficiency virus type I show increased sensitivity to neutralization by human sera. Virology, 220 (2), pp. 450-460. | Show Abstract | Read more

We constructed a number of HXB2 viruses chimeric for the gp 120 glycoprotein derived from a number of viable molecular clones obtained from a primary isolate. Comparative biological characterization of the parental primary viruses with the gp 120.HXB2 chimeras demonstrated identical patterns of cell tropism and cytopathicity. Furthermore, both parental and chimeric viruses were insensitive to neutralization by sCD4 and a panel of conformation-dependent monoclonal antibodies, demonstrating that transfer of the gp 120 protein alone was sufficient to confer a "neutralization-resistant" phenotype to the T-cell-adapted clone HXB2. We assessed the contribution of gp 120 epitopes to the neutralizing immune response by comparing the sensitivity of these viruses to neutralization by a panel of sera from HIV-infected individuals. Seven of eleven sera tested were able to neutralize HXB2 and two or more of the chimeric viruses; in contrast, only one serum neutralized more than one of the parental primary virus clones. The association of gp 120-gp41 envelope at the surface of infected PBMC cultures was measured in the presence or absence of soluble CD4. No differences in CD4-induced gp 120 dissociation were seen between the chimeric and parental virus-infected cultures. Since gp 120 conformation appeared the same between primary and chimeric viruses, we suggest that the ability of human sera to neutralize the chimeric viruses may be mediated by epitopes within gp41.

Palmer C, Balfe P, Fox D, May JC, Frederiksson R, Fenyö EM, McKeating JA. 1996. Functional characterization of the V1V2 region of human immunodeficiency virus type 1. Virology, 220 (2), pp. 436-449. | Show Abstract | Read more

The level of proviral DNA sequence variation in the V1V2 region was monitored over time in six HIV-1-infected individuals. Substitutional and length variation was observed, where the majority of length changes, ranging from 28 to 49 amino acids, was located within the V1 region. Evidence for convergent evolution in the V2 region was found. The functional significance of this variation was assessed by cloning the V1V2 sequences into an infectious molecular clone, HXB2. The majority of chimeras replicated, demonstrating that the sequences, though genetically distinct, were capable of conferring a viable phenotype. Chimeras expressing closely related sequences in a constant genetic background displayed different biological phenotypes, with respect to both cytopathicity and cell tropism. However, no association between primary V1V2 amino acid sequence and viability or cytopathicity of the chimeric virus was observed, suggesting that predictions of virus phenotype based on sequences alone may be incorrect. The effect of V1V2 variation on the overall gp 120 conformation was measured by expressing the gp 20 from a number of viable and nonviable clones. No differences were observed, suggesting that misfolding of the chimeric gp 120 protein was not an explanation for the nonviability of some virus clones. Several chimeras were noncytopathic and only able to replicate in PBMC cultures, demonstrating that the V1V2 region, independent of the V3 sequence, is capable of defining both tropism and cytopathicity.

McKeating JA, Shotton C, Jeffs S, Palmer C, Hammond A, Lewis J, Oliver K, May J, Balfe P. 1996. Immunogenicity of full length and truncated forms of the human immunodeficiency virus type I envelope glycoprotein. Immunol Lett, 51 (1-2), pp. 101-105. | Show Abstract | Read more

We have monitored the immunogenicity of a V1V2 sub-fragment of gp 120 in contrast to the full length protein and to a truncated form (PR12) where the V1, V2 and V3 regions were removed. In contrast to previously published work [1] these studies show that monomeric forms of envelope are capable of inducing antibodies specific for both linear and discontinuous epitopes. These antibodies are capable of neutralising HIV infectivity. The majority of neutralising antibodies were specific for epitopes within the V2 and V3 regions demonstrating the immunodominance of these regions in monomeric gp 120. Relatively few of the antibodies were specific for the CD4 binding site, suggesting that this region is poorly immunogenic. Immunisation of rats with the PR12 truncated protein did not significantly enhance the immunogenicity of the CD4 binding site. However, the immune response generated included antibodies capable of binding to diverse primary HIV-1 and HIV-2 envelope glycoproteins. We have shown that up to 30% of sera from HIV-1 infected individuals have antibodies that are capable of recognising conformation-dependent epitopes within the V1V2 region of the clone HXB10, suggesting the presence of conserved cross-reactive epitopes. Furthermore we have shown an association between the presence of V1V2 reactive antibodies and the neutralisation titre of the sera tested suggesting that antibodies to this region contribute to the cross-reactive neutralising response.

Shotton C, Arnold C, Sattentau Q, Sodroski J, McKeating JA. 1995. Identification and characterization of monoclonal antibodies specific for polymorphic antigenic determinants within the V2 region of the human immunodeficiency virus type 1 envelope glycoprotein. J Virol, 69 (1), pp. 222-230. | Show Abstract

We have identified six monoclonal antibodies (MAbs) mapping to both linear and conformation-dependent epitopes within the V2 region of the human immunodeficiency virus type 1 clone HXB10. Three of the MAbs (12b, 66c, and 66a) were able to neutralize the molecular clones HXB10 and HXB2, with titers in the range of 9.5 to 20.0 micrograms/ml. MAbs mapping to the crown of the V2 loop (12b, 60b, and 74) bound poorly to cell surface-expressed oligomeric gp120, suggesting an explanation for the poor or negligible neutralizing activity of MAbs to this region. In contrast, MAbs 12b and 60b demonstrated good reactivity with recombinant gp120 in an enzyme-linked immunosorbent assay format, suggesting differential epitope exposure between the recombinant and native forms of gp120. Cross-competition analysis of these MAbs and additional V1V2 MAbs for gp120 binding enabled us to assign the MAbs to six groups (A to F). Selection of neutralization escape mutants with MAbs 10/76b and 11/68b, belonging to nonoverlapping competition groups, identified amino acid changes at residues 165 (I to T) and 185 (D to N), respectively. Interestingly, these escape variants remained sensitive to neutralization by the nonselecting V2 MAbs. All MAbs demonstrated good recognition of IIIB viral gp120 yet failed to neutralize nonclonal stocks of IIIB. In addition, MAbs 12b and 62c bound MN and RF viral gp120, respectively, yet failed to neutralize the respective isolates. Cloning and expression of a library of gp120 and V1V2 fragments from IIIB-, MN-, and RF-infected H9 cultures identified a number of polymorphic sites, resulting in antigenic variation and subsequent loss of V2 MAb recognition. In contrast, the V3 region from the clones of the same isolates showed no amino acid changes, suggesting that the V2 region is polymorphic in long-term-passaged laboratory isolates and may account for the reduced antibody recognition observed.

Paul NL, Marsh M, McKeating JA, Schulz TF, Liljeström P, Garoff H, Weiss RA. 1993. Expression of HIV-1 envelope glycoproteins by Semliki Forest virus vectors. AIDS Res Hum Retroviruses, 9 (10), pp. 963-970. | Show Abstract | Read more

We have used Semliki Forest virus (SFV) vectors to express both the human immunodeficiency virus type 1 (HIV-1) envelope precursor gp160 and the cleaved external portion gp120. Expression of the foreign gene in this system is by transfection of recombinant SFV RNA, or by infection with a recombinant SFV virus that has a wide host range. pSFV1-gp120 or pSFV1-gp160 were expressed in baby hamster kidney (BHK) cells and two human cell lines: HeLa cervical carcinoma and MOLT-4 CD4+ T cells. After SFV1-gp120 infection of HeLa cells, 3.3 micrograms of gp120 was secreted into the media by 1 million cells in a 24-hr period. The secreted envelope glycoprotein was recognized by anti-gp120 monoclonal antibodies directed against both linear and conformation-dependent epitopes in different regions of the molecule. The recombinant gp120 also bound to a soluble form of the CD4 receptor. Syncytium formation was observed when MOLT-4 cells were infected with SFV1-gp160. The gp160 expressed by BHK cells induced syncytia during cocultivation with C8166 CD4+ T cells. These data indicate that SFV vectors can be used to produce the HIV-1 envelope glycoproteins to high levels, and that these proteins are correctly processed, folded, and transported to the cell surface. Furthermore, they exhibit functional activity as indicated by their ability to bind to soluble receptor and induce cell-to-cell fusion.

Klasse PJ, McKeating JA, Schutten M, Reitz MS, Robert-Guroff M. 1993. An immune-selected point mutation in the transmembrane protein of human immunodeficiency virus type 1 (HXB2-Env:Ala 582(-->Thr)) decreases viral neutralization by monoclonal antibodies to the CD4-binding site. Virology, 196 (1), pp. 332-337. | Show Abstract | Read more

An immune-selected point-mutation (HXB2-Env:Ala582(-->Thr)) in the transmembrane protein, gp41, of the human immunodeficiency virus type 1 confers relative insensitivity to neutralization by a number of sera from HIV-1-positive persons. Affinity-purified human antibodies to continuous epitopes spanning Ala582 do not neutralize the virus (C. Wilson, M. S. Reitz, Jr., K. Aldrich, P. J. Klasse, J. Blomberg, R. C. Gallo, and M. J. Robert-Guroff, J. Virol. 64, 3240-3248, 1990). The specificity of the antibodies that the mutation renders less active has not previously been determined. We now report that this substitution in gp41 reduces the neutralizing activity of monoclonal antibodies to discontinuous gp120 epitopes, which overlap with the CD4-binding site. There was no such difference in sensitivity to neutralization by soluble CD4, CD4-immunoglobulin, or by two monoclonal antibodies to the V3 region of gp120. Furthermore, the ability of 10 human HIV-1-positive sera to block the binding of soluble CD4 to mammalian-recombinant gp120 correlated weakly with their differentiation of neutralization between the wild-type and the Env:Ala582(-->Thr)-mutant virus. We thus suggest that the substitution in gp41 modulates the conformation of gp120 so as to decrease viral sensitivity to one category of antibodies which is partly responsible for the group-specific neutralization of HIV-1 by human sera.

McKeating JA, Bennett J, Zolla-Pazner S, Schutten M, Ashelford S, Brown AL, Balfe P. 1993. Resistance of a human serum-selected human immunodeficiency virus type 1 escape mutant to neutralization by CD4 binding site monoclonal antibodies is conferred by a single amino acid change in gp120. J Virol, 67 (9), pp. 5216-5225. | Show Abstract

We have selected an HXB2 variant which can replicate in the presence of a neutralizing human serum. Sequencing of the gp120 region of the env gene from the variant and parental viruses identified a single amino acid substitution in the third conserved region of gp120 at residue 375 (AGT-->AAT, Ser-->Asn; designated 375 S/N). The escape mutant was found to be resistant to neutralization by soluble CD4 (sCD4) and four monoclonal antibodies (MAbs), 39.13g, 1.5e, G13, and 448, binding to epitopes overlapping that of the CD4 binding site (CD4 b.s.). Introduction of the 375 S/N mutation into HXB2 by site-directed mutagenesis confirmed that this mutation is responsible for the neutralization-resistant phenotype. Both sCD4 and three of the CD4 b.s. MAbs (39.13g, 1.5e, and G13) demonstrated reduced binding to the native 375 S/N mutant gp120. The ability to select for an escape variant resistant to multiple independent CD4 b.s. MAbs by a human serum confirms the reports that antibodies to the discontinuous CD4 b.s. are a major component of the group-specific neutralizing activity in human sera.

McKeating JA, Shotton C, Cordell J, Graham S, Balfe P, Sullivan N, Charles M, Page M, Bolmstedt A, Olofsson S. 1993. Characterization of neutralizing monoclonal antibodies to linear and conformation-dependent epitopes within the first and second variable domains of human immunodeficiency virus type 1 gp120. J Virol, 67 (8), pp. 4932-4944. | Show Abstract

A number of linear and conformation-dependent neutralizing monoclonal antibodies (MAbs) have been mapped to the first and second variable (V1 and V2) domains of human immunodeficiency virus type 1 (HIV-1) gp120. The majority of these MAbs are as effective at neutralizing HIV-1 infectivity as MAbs to the V3 domain and the CD4 binding site. The linear MAbs bind to amino acid residues 162 to 171, and changes at residues 183/184 (PI/SG) and 191/192/193 (YSL/GSS) within the V2 domain abrogate the binding of the two conformation-dependent MAbs, 11/68b and CRA-4, respectively. Surprisingly, a change at residue 435 (Y/H or Y/S), in a region of gp120 near the CD4 binding site (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987; L. A. Lasky, G. M. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. Berman, T. Gregory, and D. Capon, Cell 50:975-985, 1987; and U. Olshevsky, E. Helseth, C. Furman, J. Li, W. Haseltine, and J. Sodroski, J. Virol. 64:5701-5707, 1990), abrogated gp120 recognition by both of the conformation-dependent MAbs. However, both MAbs 11/68b and CRA-4 were able to bind to HIV-1 V1V2 chimeric fusion proteins expressing the V1V2 domains in the absence of C4, suggesting that residues in C4 are not components of the epitopes but that amino acid changes in C4 may affect the structure of the V1V2 domains. This is consistent with the ability of soluble CD4 to block 11/68b and CRA-4 binding to both native cell surface-expressed gp120 and recombinant gp120 and suggests that the binding of the neutralizing MAbs to the virus occurs prior to receptor interaction. Since the reciprocal inhibition, i.e., antibody inhibition of CD4-gp120 binding, was not observed, the mechanism of neutralization is probably not a blockade of virus-receptor interaction. Finally, we demonstrate that linear sequences from the V2 region are immunogenic in HIV-1-infected individuals, suggesting that the primary neutralizing response may be directed to both V2 and V3 epitopes.

Klasse PJ, McKeating JA. 1993. Soluble CD4 and CD4 immunoglobulin-selected HIV-1 variants: a phenotypic characterization. AIDS Res Hum Retroviruses, 9 (7), pp. 595-604. | Show Abstract | Read more

The selection of HIV-1 resistance to neutralization by both monovalent and bivalent forms of soluble CD4 was demonstrated under various conditions. Phenotypic traits of the neutralization-resistant variants were systematically explored in order to gain insight into which aspects of the interactions with CD4 are most expendable to HIV-1 replication. The size of the nonneutralized fraction after treatment of preparations of the HIV-1 isolate IIIB and a molecular clone derived from it (HX10), with either monovalent soluble CD4 (sCD4) or bivalent CD4-Ig, was determined. These fractions were greater for the polyclonal IIIB than for the viral clone, and greater after treatment with sCD4 than with CD4-Ig. The virus in the nonneutralized fractions exhibited 2- to 20-fold lower sensitivity to the neutralizing agents than did unselected virus. In addition, clonal HIV-1 (HX10) was cultured in the presence of sCD4 or CD4-Ig for 12 weeks, so as to allow for accumulation of mutations that would confer stronger resistance to the selecting agent. Variants were obtained with up to 100-fold increased resistance to sCD4 or CD4-Ig. Detergent-solubilized gp120 from sCD4- and CD4-Ig-selected virus showed decreases in affinity for sCD4 and CD4-Ig. The monoclonal antibodies 6H10, to the gp120-binding site in domain 1 of CD4, and 5A8, to domain 2 of CD4, inhibited the induction by the viral escape variants of syncytium formation of C8166 cells. In general, the concentration of antibody 6H10 that inhibited the escape variants was lower than the concentration that inhibited the wild type, whereas there was no significant difference for the domain 2 antibody 5A8. We interpret this as a weaker attachment of the escape variants than of the wild-type virus to cellular CD4, but as an intact dependence of the variants on CD4 interactions for gaining entry into cells.

Schutten M, McKnight A, Huisman RC, Thali M, McKeating JA, Sodroski J, Goudsmit J, Osterhaus AD. 1993. Further characterization of an antigenic site of HIV-1 gp120 recognized by virus neutralizing human monoclonal antibodies. AIDS, 7 (7), pp. 919-923. | Show Abstract

OBJECTIVE: The aim of this study is to characterize antigenic sites on HIV-1 gp120 which may be important for the development of active and passive immunization strategies against HIV-1 infection. DESIGN: Two HIV-1-seropositive individuals were selected from the Amsterdam cohort and Epstein-Barr virus (EBV)-transformed B cells were generated from their peripheral blood mononuclear cells, which produce HIV-1-specific human monoclonal antibodies (HuMAb). METHODS: HuMAb were generated and selected based on their reactivities with native gp120. Reactivity with HIV-1 strains from phylogenetically different subfamilies was determined by immunostaining and virus neutralization assays. Specificity for the CD4-binding site was tested by an inhibition enzyme-linked immunosorbent assay and amino acids (aa) involved in the binding of the HuMAb were identified with a set of gp120 molecules with single aa substitutions. RESULTS: Three HuMAb (GP13, GP44, GP68) were generated, all recognizing a conserved conformation dependent epitope within, or topographically near, the CD4-binding site of gp120. HuMAb GP13 and GP68 neutralized a broad range of HIV-1 strains from phylogenetically different subfamilies, whereas HuMAb GP44 exhibited a more restricted pattern of neutralizing activity. The patterns of gp120 aa involved in their binding were unique for each of these HuMAb. CONCLUSIONS: The pattern of reactivities of these three HIV-1-neutralizing HuMAb developed in these studies is similar to, but distinct from other human and rodent MAb that recognize this antigenic site of HIV-1 gp120.

McKeating JA, Cordell J, Dean CJ, Balfe P. 1992. Synergistic interaction between ligands binding to the CD4 binding site and V3 domain of human immunodeficiency virus type I gp120. Virology, 191 (2), pp. 732-742. | Show Abstract | Read more

We demonstrate that soluble CD4 (sCD4) or a monoclonal antibody (mAb), 39.13g, binding to a conformational epitope of gp120 involved in CD4 binding, and mAbs binding to the V3 domain of gp120, can synergistically neutralize human immunodeficiency virus type I (HIV-1). In contrast, a neutralizing mAb binding to a linear epitope within the CD4 binding domain was unable to exert a synergistic effect in combination with V3 mAbs, suggesting that synergism is dependent on ligands binding to the critical, discontinuous, gp120 residues constituting the CD4 binding site. A number of V3 mAbs showed increased binding to virion gp120 in the presence of sCD4, suggesting a mechanism for the synergistic neutralization. This effect was not observed with recombinant or detergent solubilized viral gp120, suggesting that the oligomeric structure of gp120 on viral particles affects V3 epitope exposure. This hypothesis is supported by the ability of two new V3 mAbs, 8/38c and 8/64b, to only neutralize HIV-1 in the presence of sCD4 or mAb 39.13g; binding studies demonstrate that these mAbs only bind to virion gp120 in the presence of sCD4. Thus, V3 epitope exposure is modulated by the interaction of virion gp120 with ligands specific for the CD4 binding domain and results in enhanced antibody-mediated neutralization.

McKeating JA, Thali M, Furman C, Karwowska S, Gorny MK, Cordell J, Zolla-Pazner S, Sodroski J, Weiss RA. 1992. Amino acid residues of the human immunodeficiency virus type I gp120 critical for the binding of rat and human neutralizing antibodies that block the gp120-sCD4 interaction. Virology, 190 (1), pp. 134-142. | Show Abstract | Read more

We have characterized the discontinuous epitopes recognized by two rat and three human neutralizing monoclonal antibodies (mAb) by examining the effect of single amino acid changes in conserved residues of gp120 on mAb recognition. A human mAb derived from an infected individual, 448D, and two rat mAbs, 39.13g and 39.3b, respectively, derived by immunization with native recombinant gp120, recognize similar epitopes. Recognition of the envelope glycoproteins by these mAbs was affected by changes in gp120 amino acid residues 88, 113, 117, 257, 368, or 370. The gp120 amino acids 257, 368, and 370 have previously been reported to be important for CD4 binding, which is consistent with the ability of these mAbs to block the gp120-CD4 interaction. Residues 88, 113, and 117 are not thought to be important for CD4 binding, suggesting that the antibody epitopes overlap, but are distinct from, the CD4 binding region. We also found that some alterations in gp120 residues 88, 117, 368, or 421 reduced the ability of polyclonal sera from HIV-1-infected individuals to inhibit the interaction of the mutant gp120 glycoproteins with soluble CD4. Thus, changes in the HIV-1 gp120 glycoprotein that minimally affect the receptor binding may allow escape from neutralizing antibodies directed against the CD4 binding region.

O'Hagan DT, Rafferty D, McKeating JA, Illum L. 1992. Vaginal immunization of rats with a synthetic peptide from human immunodeficiency virus envelope glycoprotein. J Gen Virol, 73 ( Pt 8) (8), pp. 2141-2145. | Show Abstract | Read more

Local secretory immunity in the vagina may confer a degree of protection against heterosexual transmission of human immunodeficiency virus (HIV). Since the vagina has been shown to respond to local immunization, we have undertaken intravaginal immunization of rats with a 20-mer peptide (amino acid residues 102 to 121) of the HIV-1 envelope glycoprotein (gp120). The peptide was administered in combination with an 'absorption enhancer', lysophosphatidyl glycerol (LPG), which has previously been shown to promote the absorption of intravaginally administered peptides, while exerting only mild effects on epithelial membrane integrity. Intravaginal immunization with LPG and the peptide induced serum and vaginal wash IgA and IgG antibody responses which were enhanced in comparison to those after immunization with the peptide alone. Serum antibodies induced by both subcutaneous and intravaginal immunization were able to recognize recombinant HIV-1 gp120. However, the rat antiserum displayed no neutralizing activity against the virus. These results demonstrate that LPG is an effective immunological adjuvant for intravaginally administered peptide antigens. An alternative absorption enhancer, bestatin (BES), was not effective as an immunological adjuvant when administered intravaginally and blocked the adjuvant activity of LPG when BES and LPG were used in combination.

Moore JP, Simpson G, McKeating JA, Burakoff SJ, Schreiber SL, Weiss RA. 1992. CPF-DD is an inhibitor of infection by human immunodeficiency virus and other enveloped viruses in vitro. Virology, 188 (2), pp. 537-544. | Show Abstract | Read more

The initial step in the infection cycle of human immunodeficiency virus type 1 (HIV-1) involves binding of its surface glycoprotein gp 120 to the T lymphocyte CD4 antigen. CPF-DD is a low molecular weight inhibitor of HIV infectivity that inhibits gp 120 binding to CD4 in vitro (Finberg et al., Science 249, 287-291, 1990). We find, however, that the actions of CPF-DD are not limited to its ability to interfere with gp 120-CD4 binding; its predominant action is to remove the viral envelope from the underlying core. Subsequently the virions disintegrate. Most enveloped viruses tested were inhibited by CPF-DD, but the infectivity of noneneloped viruses was unaffected or only slightly reduced.

McKeating JA. 1992. CD4 antagonists. Biochem Soc Trans, 20 (2), pp. 521-525. | Read more

McKeating JA, Moore JP, Ferguson M, Marsden HS, Graham S, Almond JW, Evans DJ, Weiss RA. 1992. Monoclonal antibodies to the C4 region of human immunodeficiency virus type 1 gp120: use in topological analysis of a CD4 binding site. AIDS Res Hum Retroviruses, 8 (4), pp. 451-459. | Show Abstract | Read more

We have raised antisera and monoclonal antibodies (MAbs) to the C4 region of HIV-1 gp120, using an antigen chimaera of poliovirus as immunogen. These MAbs and sera, together with MAbs to the same region raised by other methods, fall into three groups defined by their abilities to bind to recombinant gp120 and/or the immunogenic peptide. In some cases, the amino acids recognized by the MAbs have been identified by pep-scan and by solution phase peptide inhibition of binding to recombinant gp120. Our results indicate that the amino acids WQEVGKAMYA are exposed on the surface of recombinant gp120. Antibodies to these amino acids on recombinant gp120 compete for soluble CD4 binding in vitro, but only weakly neutralize HIV.

Mckeating JA. 1992. Neutralization of human immunodeficiency virus Reviews in Medical Virology, 2 (1), pp. 35-42. | Read more

Moore JP, McKeating JA, Huang YX, Ashkenazi A, Ho DD. 1992. Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates. J Virol, 66 (1), pp. 235-243. | Show Abstract

Primary isolates of human immunodeficiency virus type 1 (HIV-1) are much less sensitive to neutralization by soluble CD4 (sCD4) and sCD4-immunoglobulin (Ig) chimeras (CD4-IgG) than are HIV-1 strains adapted to growth in cell culture. We demonstrated that there are significant reductions (10- to 30-fold) in the binding of sCD4 and CD4-IgG to intact virions of five primary isolates compared with sCD4-sensitive, cell culture-adapted isolates RF and IIIB. However, soluble envelope glycoproteins (gp120) derived from the primary isolate virions, directly by detergent solubilization or indirectly by recombinant DNA technology, differed in affinity from RF and IIIB gp120 by only one- to threefold. The reduced binding of sCD4 to these primary isolate virions must therefore be a consequence of the tertiary or quaternary structure of the envelope glycoproteins in their native, oligomeric form on the viral surface. In addition, the rate and extent of sCD4-induced gp120 shedding from these primary isolates was lower than that from RF. We suggest that reduced sCD4 binding and increased gp120 retention together account for the relative resistance of these primary isolates to neutralization by sCD4 and CD4-IgG and that virions of different HIV-1 isolates vary both in the mechanism of sCD4 binding and in subsequent conformational changes in their envelope glycoproteins.

Cordell J, Moore JP, Dean CJ, Klasse PJ, Weiss RA, McKeating JA. 1991. Rat monoclonal antibodies to nonoverlapping epitopes of human immunodeficiency virus type 1 gp120 block CD4 binding in vitro. Virology, 185 (1), pp. 72-79. | Show Abstract | Read more

Monoclonal antibodies (MAbs) to a recombinant form of the envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1 IIIB) were raised in rats and screened for their ability to block recombinant gp120 binding to recombinant, soluble CD4 (sCD4) in vitro. Four such MAbs were identified and characterised. Each MAb bound strongly to gp120 from eight widely divergent HIV-1 strains from the United States and Africa. Two MAbs were mapped to the fourth conserved (C4) region of gp120, whereas the other two recognised an as yet undefined, conformationally sensitive epitope. MAbs to the latter epitope were the more potent in blocking the gp120-sCD4 interaction. None of the MAbs, however, had potent neutralising activity.

Moore JP, McKeating JA, Norton WA, Sattentau QJ. 1991. Direct measurement of soluble CD4 binding to human immunodeficiency virus type 1 virions: gp120 dissociation and its implications for virus-cell binding and fusion reactions and their neutralization by soluble CD4. J Virol, 65 (3), pp. 1133-1140. | Show Abstract

We have analyzed the binding of soluble CD4 (sCD4) to human immunodeficiency virus type 1 (HIV-1) virions (isolates IIIB and RF) at 4 and 37 degrees C by using a combination of gel exclusion chromatography and enzyme-linked immunosorbent assay detection systems. The sCD4 binding curve at 37 degrees C indicates that the affinity of the interaction of sCD4 with gp120 on the virion surface is indistinguishable from the affinity of sCD4 for the equivalent concentration of soluble gp120. At 4 degrees C, however, the affinity of sCD4 for virion-bound gp120 but not for soluble gp120 is reduced by about 20-fold. Binding of sCD4 (greater than 0.2 microgram/ml) to virions at 37 degrees C but not 4 degrees C induces the rapid dissociation of a major proportion of gp120 from gp41 on the virion surface. This dissociation requires occupancy by sCD4 of multiple (probably two) binding sites on a gp120-gp41 oligomer. At 37 degrees C there are two components to the neutralizing action of sCD4 on HIV-1; reversible, competitive inhibition at low sCD4 concentrations (less than 0.2 microgram/ml) and essentially irreversible inhibition due to gp120 loss at higher sCD4 concentrations. At 4 degrees C, sCD4 neutralizes HIV infectivity by competitive inhibition alone. These findings may have implications for the HIV-CD4+ cell binding and fusion reactions and the mechanism by which sCD4 blocks infectivity.

McKeating JA, Moore JP. 1991. HIV infectivity. Nature, 349 (6311), pp. 660. | Read more

McKeating JA, McKnight A, Moore JP. 1991. Differential loss of envelope glycoprotein gp120 from virions of human immunodeficiency virus type 1 isolates: effects on infectivity and neutralization. J Virol, 65 (2), pp. 852-860. | Show Abstract

Several parameters which may affect the infectivity of human immunodeficiency virus type 1 in tissue culture were analyzed. In particular, we used gel exclusion chromatography to investigate how the loss of the surface glycoprotein gp120 from virions of the HTLV-IIIB (IIIB), HTLV-IIIRF (RF), and SF-2 isolates modulates infectivity. In IIIB and RF cultures, a high proportion of the total gp120 was virion bound initially but was gradually lost from the virions over time. In contrast, most of the gp120 (and p24) in SF-2-infected cultures was soluble and the few particles present had a fivefold-lower level of virus-bound gp120. However, this reduced level of virion-bound gp120 was more resistant to shedding. Loss of a major proportion of gp120 from IIIB and RF virions resulted in reduced infectivities, and in addition, the resulting accumulation of soluble gp120 in the cultures could competitively inhibit viral infection, especially with SF-2. Increased shedding of virion gp120 also affected the neutralization of IIIB and RF particles. However, the high sensitivity to human serum neutralization characteristic of SF-2 was unaffected by soluble gp120 in cultures, suggesting that the epitopes responsible are not present on soluble gp120.

Steimer KS, Klasse PJ, McKeating JA. 1991. HIV-1 neutralization directed to epitopes other than linear V3 determinants. AIDS, 5 Suppl 2 pp. S135-S143.

Clements GJ, Price-Jones MJ, Stephens PE, Sutton C, Schulz TF, Clapham PR, McKeating JA, McClure MO, Thomson S, Marsh M. 1991. The V3 loops of the HIV-1 and HIV-2 surface glycoproteins contain proteolytic cleavage sites: a possible function in viral fusion? AIDS Res Hum Retroviruses, 7 (1), pp. 3-16. | Show Abstract | Read more

Located close to the crown of the V3 type-specific neutralization loop of the human immunodeficiency virus type 1 (HIV-1) (IIIB) SU glycoprotein gp120, are several potential sites that should be susceptible to proteolytic cleavage by enzymes of trypsinlike or chymotrypsinlike specificity, or by aspartic proteinases. The linkages potentially sensitive to chymotryptic/aspartic proteinase cleavage are retained also within the equivalent domain of HIV-2 (ROD) gp105. We show that thrombin and tryptase cleave HIV-1 gp120 specifically at the tryptic site (GPGR decreases AFVT), and that cathepsin E, an endosomal aspartic proteinase, cleaves at the chymotrypsinlike site (GPGRAF decreases VT). HIV-2 gp105 is also cut by cathepsin E at a site (QIML decreases MSGH) in its V3 loop. Cleavage of HIV-1 gp120 by thrombin is enhanced by sCD4 binding, but is prevented by transient exposure of gp120 to nonionic detergent. Thrombin treatment of HIV-1 gp120 destroys the binding sites for some neutralizing monoclonal antibodies (MAbs) on the V3 loop, but does not affect the affinity of gp120 for sCD4. Conversely, binding of neutralizing MAbs to the HIV-1 V3 loop prior to addition of thrombin or cathepsin E blocks the cleavage reactions, and the binding of some HIV-positive sera to gp120 blocks thrombin cleavage. Analysis of published sequences suggests that all HIV-1, HIV-2, and simian immunovirus (SIV) isolates contain potential proteolytic cleavage sites at similar positions in their V3 loops or equivalent domains. We suggest that cleavage of the V3 loop by a cell surface or endosomal proteinase occurs during the HIV-cell fusion reaction, and that neutralizing antibodies directed against the V3 loop might act by inhibition of this reaction.

Ho DD, McKeating JA, Li XL, Moudgil T, Daar ES, Sun NC, Robinson JE. 1991. Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody. J Virol, 65 (1), pp. 489-493. | Show Abstract

A human monoclonal antibody designated 15e is reactive with the envelope glycoprotein (gp120) of multiple isolates of human immunodeficiency virus type 1 (HIV-1). Antibody 15e also neutralizes HIV-1 with broad specificity and blocks gp120 binding to CD4. Characterization of the 15e epitope shows that it is conformation dependent and is distinct from previously recognized functional domains of gp120, suggesting that this epitope represents a novel site important for HIV-1 neutralization and CD4 binding. These findings have implications for the development of a vaccine for AIDS.

Moore JP, McKeating JA, Weiss RA, Sattentau QJ. 1990. Dissociation of gp120 from HIV-1 virions induced by soluble CD4. Science, 250 (4984), pp. 1139-1142. | Show Abstract | Read more

The CD4 antigen is the high affinity cellular receptor for the human immunodeficiency virus type-1 (HIV-1). Binding of recombinant soluble CD4 (sCD4) or the purified V1 domain of sCD4 to the surface glycoprotein gp120 on virions resulted in rapid dissociation of gp120 from its complex with the transmembrane glycoprotein gp41. This may represent the initial stage in virus-cell and cell-cell fusion. Shedding of gp120 from virions induced by sCD4 may also contribute to the mechanism by which these soluble receptor molecules neutralize HIV-1.

Moore JP, McKeating JA, Jones IM, Stephens PE, Clements G, Thomson S, Weiss RA. 1990. Characterization of recombinant gp120 and gp160 from HIV-1: binding to monoclonal antibodies and soluble CD4. AIDS, 4 (4), pp. 307-315. | Show Abstract | Read more

We compared four preparations of recombinant HIV-1 envelope glycoprotein: mammalian (Chinese hamster ovary cells) gp120 (Celltech); baculovirus gp120 from American Biotechnologies Inc. (ABT) and from MicroGeneSys (MGS); and baculovirus gp160 (Institute of Virology, Oxford, UK). Each envelope glycoprotein binds to a neutralizing monoclonal antibody (MAb) directed against the V3 loop, confirming the integrity of this type-specific neutralization epitope. MGS gp120 binds abnormally well to a MAb which recognizes an epitope preferentially exposed on denatured gp120. Consistent with this finding, MGS gp120 binds to soluble CD4 (sCD4) with an affinity 50-100-fold lower than that of Celltech gp120. The affinity of Celltech gp120 from sCD4 is 2.3 nM, indistinguishable from that of gp120 extracted from HIV-1 virions. Baculovirus gp120 (ABT) and gp160 also have a high affinity for sCD4. A significant proportion of anti-gp120 antibodies in HIV-positive human sera recognize epitopes that are dependent on the mammalian glycosylation pattern, and a human HIV-positive serum inhibits the binding of mammalian gp120 to sCD4 five- to 10-fold more potently than it inhibits baculovirus gp120 binding to sCD4.

McKeating JA, Griffiths PD, Weiss RA. 1990. HIV susceptibility conferred to human fibroblasts by cytomegalovirus-induced Fc receptor. Nature, 343 (6259), pp. 659-661. | Show Abstract | Read more

The main receptor for the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) on T and B lymphocytes, monocytes and macrophages is the CD4 antigen 1-3. Infection of these cells is blocked by monoclonal antibodies to CD4(1,2) and by recombinant soluble CD4(4-9). Expression of transfected CD4 on the surface of HeLa and other human cells renders them susceptible to HIV infection 10. HIV-antibody complexes can also infect monocytes and macrophages by means of receptors for the Fc portion of immunoglobulins (FcR)11-13), or complement receptors 14,15. The expression of IgG FcRs can be induced in cells infected with human herpes viruses such as herpes simplex virus type 1 (HSV-1)16,17 and human cytomegalovirus (CMV)18-21. Here we demonstrate that FcRs induced by CMV allow immune complexes of HIV to infect fibroblasts otherwise not permissive to HIV infection. Infection was inhibited by prior incubation with human IgG, but not by anti-CD4 antibody or by recombinant soluble CD4. Once HIV had entered CMV-infected cells by means of the FcR, its replication could be enhanced by CMV transactivating factors. Synergism between HIV and herpes viruses could also operate in vivo, enhancing immunosuppression and permitting the spread of HIV to cells not expressing CD4.

Putney SD, McKeating JA. 1990. Antigenic variation in HIV. AIDS, 4 Suppl 1 (SUPPL. 1), pp. S129-S136.

McKeating JA, Gow J, Goudsmit J, Pearl LH, Mulder C, Weiss RA. 1989. Characterization of HIV-1 neutralization escape mutants. AIDS, 3 (12), pp. 777-784. | Show Abstract | Read more

Infection by molecularly cloned HIV-1, in the presence of a high-titre neutralizing monoclonal antibody (MAb), resulted in the selection of plaques in MT4 cells releasing HIV resistant to neutralization by the same MAb. The epitope recognized by the MAb was mapped to the V3 neutralization epitope at amino acids 305-321. The HIV-1 variants showed a reduced binding capacity for the selecting MAb as determined by immunofluorescence. Polymerase chain reaction (PCR) amplification of complementary DNA derived from viral RNA, cloning and sequencing identified a base pair (bp) change C----G at position 6663 in variant 110.5/1, predicting a change at amino acid 308 Arg----Gly. No other changes in the epitope were observed by sequencing three other variants. Differential hybridization of PCR amplified viral RNA and DNA, with oligonucleotides specific for the observed bp change or the 'wild type' sequence, indicated that the variants 110.5/1 and 110.5/7 were genotypically mixed for 308Gly/Arg. Subsequent screening of biologically 'recloned' variants 110.5/1 and 110.5/7 identified two subclones homozygous for the 308Gly change. The Arg----Gly change appears to affect the binding of the antibody to the epitope, since the linear peptide substituting 308Gly for 'wild type' 308Arg was 100 times less potent in blocking the neutralization of parental HIV. Amino-acid residue 308 thus appears to be crucial for antibody binding to the epitope. In addition, mutations distant from the monoclonal antibody binding site may also affect neutralization by antibodies recognizing the V3 loop.

McKeating JA, McKnight A, McIntosh K, Clapham PR, Mulder C, Weiss RA. 1989. Evaluation of human and simian immunodeficiency virus plaque and neutralization assays. J Gen Virol, 70 ( Pt 12) (12), pp. 3327-3333. | Show Abstract | Read more

A number of CD4+ T cell lines were compared for their ability to act as target cells for human immunodeficiency virus (HIV) infection in syncytium- and plaque-forming assays. MT-4 and C8166 cells were the most sensitive indicator cells for HIV- and simian immunodeficiency virus (SIV)-induced cytopathic effects, and gave rise to macroscopic (MT-4) and microscopic (C8166) plaques. The MT-4 plaque assay was evaluated for the measurement of HIV- and SIV-neutralizing antibodies.

Moore JP, Wallace LA, Follett EA, McKeating JA. 1989. An enzyme-linked immunosorbent assay for antibodies to the envelope glycoproteins of divergent strains of HIV-1. AIDS, 3 (3), pp. 155-163. | Show Abstract

We have developed an enzyme-linked immunosorbent assay (ELISA) specific for antibodies to the envelope glycoproteins gp120 and gp160 of HIV-1. An antibody to a conserved epitope on gp120 is adsorbed to a solid phase and used to capture gp120 and/or gp160 from solution. This may be purified recombinant protein or in simple, non-denaturing detergent extracts of different strains of HIV-1. Human serum antibodies bound to the captured antigen are subsequently detected with an anti-human antibody conjugated to alkaline phosphatase, and the AMPAK ELISA amplification system (Novo BioLabs, Cambridge, UK). With this procedure, antibodies can be detected that recognize gp120 from a wide range of divergent HIV-1 strains. The ELISA is sufficiently sensitive to detect env antibodies in sera from HIV-positive individuals at dilutions of 1:300,000. No repeatable false-positives were detected in a screen of 250 normal serum samples. Env antibodies were detected in all 37 strongly HIV-positive sera tested, and in four sera that were borderline or weakly positive in commercial ELISA. However, 55 sera positive in commercial ELISA but unconfirmable by Western blot ('ambiguously' positive) did not contain detectable env antibodies.

McKeating JA, Willey RL. 1989. Structure and function of the HIV envelope. AIDS, 3 Suppl 1 (SUPPL. 1), pp. S35-S41.

McClure MO, Schulz TF, Tedder RS, Gow J, McKeating JA, Weiss RA, Baskerville A. 1989. Inoculation of New World primates with the human immunodeficiency virus. J Med Primatol, 18 (3-4), pp. 329-335. | Show Abstract

The susceptibility of common marmosets and cotton-top tamarins to infection by HIV-2 in vivo was tested. One year and 19 months, respectively, post-inoculation, sera taken from three of four animals from each species are reactive for HIV-2 antibodies and HIV-specific nucleotide sequences were demonstrated in short-term cultures of PBL from two cotton-top tamarins. The animals remain in good health.

Grundy JE, Ayles HM, McKeating JA, Butcher RG, Griffiths PD, Poulter LW. 1988. Enhancement of class I HLA antigen expression by cytomegalovirus: role in amplification of virus infection. J Med Virol, 25 (4), pp. 483-495. | Show Abstract | Read more

We have investigated the effect of cytomegalovirus (CMV) infection on the expression of class I HLA antigens on fibroblasts in vitro. Scanning and integrating microdensitometry was used to quantitate the level of cytoplasmic class I antigen expression, and an antibody binding assay was used to quantitate cell surface expression of class I HLA molecules. CMV infection resulted in a significant increase in the level of cytoplasmic and cell surface class I HLA antigen expression of fibroblast monolayers. The maximal effect was seen at 72 hours postinfection and was observed with both the laboratory strain of CMV, strain AD169, and with CMV purified directly from clinical specimens. Part of the increased HLA expression was mediated by interferon released from infected cells; however, an additional direct effect of the virus itself has not been ruled out. Interferon-induced increased expression of class I HLA antigens was accompanied by increased binding of CMV to the cells, consistent with our recent demonstration that class I HLA molecules can function as a cellular receptor for CMV.

Grundy JE, McKeating JA, Sanderson AR, Griffiths PD. 1988. Cytomegalovirus and beta 2 microglobulin in urine specimens. Reciprocal interference in their detection is responsible for artifactually high levels of urinary beta 2 microglobulin in infected transplant recipients. Transplantation, 45 (6), pp. 1075-1079. | Show Abstract

We have previously shown that the binding of host beta 2 microglobulin (beta 2 m) by cytomegalovirus (CMV) in body fluids masks the viral antigenic determinants preventing its detection by CMV-specific monoclonal antibodies. We now report that the recognition of CMV-bound beta 2m by anti-beta 2m-specific antibodies in assays for beta 2m, results in erroneously high readings for beta 2m levels in urine specimens containing CMV. Urinary beta 2m levels have previously been reported to be elevated in patients with CMV infection--however, when virion bound beta 2m was removed by ultracentrifugation of urine specimens, the levels of free beta 2m were not found to be elevated in these patients. Since CMV is frequently excreted by transplant recipients and acquired immunodeficiency syndrome patients, our data suggest that measurements of urinary beta 2m levels in such patients are unreliable unless the urine specimens are confirmed to be free of CMV before analysis.

Weiss RA, Clapham PR, McClure MO, McKeating JA, McKnight A, Dalgleish AG, Sattentau QJ, Weber JN. 1988. Human immunodeficiency viruses: neutralization and receptors. J Acquir Immune Defic Syndr, 1 (6), pp. 536-541. | Show Abstract

The envelope glycoproteins of HIV, gp120 and gp41, contain epitopes recognized by neutralizing antibodies. Studies of human sera from infected individuals indicate that group-specific neutralization antigens common to most isolates of HIV-1 exist, and that some HIV-2 antisera cross-neutralize HIV-1. Neutralization epitopes for HIV-1 have been identified and mapped, including a group-specific antigen on gp41, and a type-specific antigen on gp120. Neutralization "escape" mutants have been selected in vitro with a neutralizing mab to the type-specific antigenic loop. The CD4 antigen binds HIV-1 gp120 with high affinity and acts as the receptor on human and simian T-lymphocytes and monocytes for all strains of HIV-1, HIV-2, and SIV tested. Following binding to the CD4 receptor, HIV becomes internalized by a pH-independent process. The principle binding domain for gp120 is located in the N-terminal V domain of CD4. Anti-idiotypic sera to CD4 mabs recognizing the same site weakly neutralize HIVs of many strains, and soluble, recombinant forms of CD4 strongly neutralize HIV. Neither anti-CD4 mabs nor sCD4 inhibit the low level of plating of HIV observed on tumour cells in culture of glial (brain) and muscle origin, indicating that CD4 is not essential for infection of these cell types.

Grundy JE, McKeating JA, Ward PJ, Sanderson AR, Griffiths PD. 1987. Beta 2 microglobulin enhances the infectivity of cytomegalovirus and when bound to the virus enables class I HLA molecules to be used as a virus receptor. J Gen Virol, 68 ( Pt 3) (3), pp. 793-803. | Show Abstract | Read more

We have previously demonstrated that human cytomegalovirus (CMV) binds the host protein beta 2 microglobulin (beta 2m) from body fluids or from cell culture media. In this report we have examined the effect of the beta 2m on viral infectivity. We have shown that the addition of human purified beta 2m, or a fraction of foetal calf serum corresponding to bovine beta 2m, to culture medium increased the amount of infectious extracellular CMV, compared to that from cells grown in serum-free medium. Metabolic labelling experiments demonstrated that this effect was not due to an increase in the amount of extracellular virus but to an increase in the infectivity of the virus present in extracellular fluids. We concluded that the binding of beta 2m by CMV increased its infectivity. We have shown that CMV and beta 2m compete for binding sites on fibroblasts. As the main binding site on cells for beta 2m is the class I HLA heavy chain we compared the binding of CMV to the Raji and Daudi cell lines which express or lack expression of class I HLA molecules. The binding of radiolabelled beta 2m-coated CMV was significantly higher to Raji cells than to Daudi cells. Furthermore, CMV could compete with beta 2m for binding to Raji cells, although the reverse was not true. These results demonstrate that CMV can use class I HLA molecules as a virus receptor. We propose that when coated with beta 2m, CMV has the capacity to displace beta 2m from the class I HLA heavy chain-beta 2m dimer on the cell surface and bind to cells. The fact that beta 2m enhances infectivity suggests that such binding leads to productive infection of cells.

Grundy JE, McKeating JA, Griffiths PD. 1987. Cytomegalovirus strain AD169 binds beta 2 microglobulin in vitro after release from cells. J Gen Virol, 68 ( Pt 3) (3), pp. 777-784. | Show Abstract | Read more

We previously reported that a host protein, beta 2 microglobulin (beta 2m) inhibited the detection of human cytomegalovirus (CMV) in urine specimens by enzyme immunoassay and postulated that beta 2m bound to the virus particle and masked the viral antigenic determinants. We report here that CMV strain AD169 grown in cell culture bound human beta 2m when this protein was added to cell culture fluids or when the virus was added to urine. Such binding was not seen with herpes simplex virus. CMV could also bind bovine beta 2m from foetal calf serum in cell culture fluids. The use of radiolabelled beta 2m in other experiments showed that CMV bound beta 2m after release from cells and that the bound beta 2m did not represent acquisition of class I HLA molecules during budding from host cell membranes. Immunoprecipitation studies showed that beta 2m was bound by two viral envelope proteins beta 2m BP1 (beta 2m-binding protein 1) and beta 2m BP2 of molecular masses 36,000 and 65,000 daltons respectively. beta 2m could not bind to separated viral proteins under reducing or non-reducing conditions. We propose that interaction of these two proteins on the viral surface is required to enable CMV to bind beta 2m.

McKeating JA, Griffiths PD, Grundy JE. 1987. Cytomegalovirus in urine specimens has host beta 2 microglobulin bound to the viral envelope: a mechanism of evading the host immune response? J Gen Virol, 68 ( Pt 3) (3), pp. 785-792. | Show Abstract | Read more

We have previously reported that human cytomegalovirus (CMV) from urine specimens cannot be captured onto a solid phase by CMV-specific monoclonal antibodies that can capture CMV grown in vitro. We report here that CMV exists in vivo in body fluids such as urine as beta 2 microglobulin (beta 2 m)-coated particles. We have demonstrated the presence of beta 2m on CMV purified directly from urine by Western blotting and have shown that the beta 2m was associated with the viral envelope. Urinary CMV could be specifically bound by an affinity column comprising a monoclonal antibody specific for beta 2m bound to Sepharose. The beta 2m-coated urinary CMV could not be neutralized by hyperimmune globulin, human immune sera or murine monoclonal antibodies that could neutralize CMV grown in cell culture. We conclude that the binding of beta 2m by CMV masks the important antigenic sites necessary for neutralization which are recognized by man's immune response. We propose that CMV has evolved this mechanism of coating itself in a host protein as a mechanism of evading the host immune response and facilitating transmission between individuals.

McKeating JA, Grundy JE, Varghese Z, Griffiths PD. 1986. Detection of cytomegalovirus by ELISA in urine samples is inhibited by beta 2 microglobulin. J Med Virol, 18 (4), pp. 341-348. | Show Abstract | Read more

During development of an enzyme immunoassay for the detection of cytomegalovirus (CMV) we previously discovered that virus found naturally in urine specimens could not be captured onto the solid phase by CMV-specific monoclonal antibodies, whereas these same antibodies could capture CMV from cell culture supernatants. We now report that urine from normal CMV-seronegative individuals contains a substance of molecular weight 11-12,000 daltons that inhibits the ELISA detection of cell culture-grown CMV. The addition of a known urinary protein of this molecular weight, beta 2 microglobulin (beta 2m; 11,700 daltons), inhibited the detection of cell culture-grown CMV in the ELISA over the concentration range found in clinical urine samples. In contrast, another low molecular weight urinary protein, lysozyme, had no inhibitory effect. beta 2m caused inhibition only when added to the virus preparation and not to the antibody-capture stage. We conclude that beta 2m in urine prevents the detection of CMV by ELISA by binding to the virus and masking its antigenic determinants and we calculate that of the order of 10(5) molecules of beta 2m bind to each particle of cell culture-grown CMV. We postulate that CMV in fresh urine specimens is similarly coated with beta 2m, accounting for the failure to detect it by ELISA.

McKeating JA, Stagno S, Stirk PR, Griffiths PD. 1985. Detection of cytomegalovirus in urine samples by enzyme-linked immunosorbent assay. J Med Virol, 16 (4), pp. 367-373. | Show Abstract | Read more

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytomegalovirus (CMV) in urine using monoclonal antibodies directed against CMV as a capture for viral antigen. The assay was capable of detecting virus at 10(2.3)TCID50/ml as determined by titration of stock virus, strain Ad169. The assay was found to have a sensitivity of 65% and a specificity of 100% when 73 coded stored urine specimens were examined. Assuming that the poor sensitivity was due to loss of antigen following storage, we proceeded to analyse fresh urine specimens. Surprisingly, the assay gave negative results with 46 fresh urines known to contain CMV; however, following storage at +4 degrees C for two weeks, 35 (76%) of these samples gave ELISA results in the positive range. This detection of CMV, after storage at +4 degrees C, could be due to degradation of virus particles leading to release of soluble glycoproteins into the medium or to the presence of an inhibitory substance in fresh urine that is destroyed during storage.

McKeating JA, Al-Nakib W, Greenaway PJ, Griffiths PD. 1985. Detection of cytomegalovirus by DNA-DNA hybridization employing probes labelled with 32-phosphorus or biotin. J Virol Methods, 11 (3), pp. 207-216. | Show Abstract | Read more

Various factors influencing the detection of human cytomegalovirus (HCMV) in infected cells by DNA-DNA hybridization have been investigated. Employing the Hind III O fragment of HCMV AD169 labelled with 32P, we found that detection sensitivity was highly influenced by the method employed for extraction of DNA from infected cells. Excision of the Hind III O fragment from the vector by restriction endonuclease digestion prior to 32P-labelling further improved the detection capability of the probe. Similarly, cytomegalovirus (CMV) DNA detection employing biotin-labelled probes and streptavidin/alkaline phosphatase in the hybridot assay was also highly dependent on the method of DNA extraction prior to hybridization. Finally, we describe an in situ assay employing a biotin-labelled probe and fluorescein-conjugated avidin to detect CMV DNA in cultured cells.

Zhuang X, Rambhatla SB, Lai AG, McKeating JA. 2017. Interplay between circadian clock and viral infection. J Mol Med (Berl), 95 (12), pp. 1283-1289. | Show Abstract | Read more

The circadian clock underpins most physiological conditions and provides a temporal dimension to our understanding of body and tissue homeostasis. Disruptions of circadian rhythms have been associated with many diseases, including metabolic disorders and cancer. Recent literature highlights a role for the circadian clock to regulate innate and adaptive immune functions that may prime the host response to infectious organisms. Viruses are obligate parasites that rely on host cell synthesis machinery for their own replication, survival and dissemination. Here, we review key findings on how circadian rhythms impact viral infection and how viruses modulate molecular clocks to facilitate their own replication. This emerging area of viral-clock biology research provides a fertile ground for discovering novel anti-viral targets and optimizing immune-based therapies.

Fletcher NF, Wilson GK, Murray J, Hu K, Lewis A, Reynolds GM, Stamataki Z, Meredith LW, Rowe IA, Luo G et al. 2012. Hepatitis C virus infects the endothelial cells of the blood-brain barrier. Gastroenterology, 142 (3), pp. 634-643.e6. | Show Abstract | Read more

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS. METHODS: We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells by using quantitative polymerase chain reaction and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture-derived HCV were used to study the ability of endothelial cells to support viral entry and replication. RESULTS: Using quantitative polymerase chain reaction, we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hCMEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor BI, and claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis. CONCLUSIONS: Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies.

Lupberger J, Zeisel MB, Xiao F, Thumann C, Fofana I, Zona L, Davis C, Mee CJ, Turek M, Gorke S et al. 2011. EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nat Med, 17 (5), pp. 589-595. | Show Abstract | Read more

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.

Harris HJ, Davis C, Mullins JGL, Hu K, Goodall M, Farquhar MJ, Mee CJ, McCaffrey K, Young S, Drummer H et al. 2010. Claudin association with CD81 defines hepatitis C virus entry. J Biol Chem, 285 (27), pp. 21092-21102. | Show Abstract | Read more

Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.

Timpe JM, Stamataki Z, Jennings A, Hu K, Farquhar MJ, Harris HJ, Schwarz A, Desombere I, Roels GL, Balfe P, McKeating JA. 2008. Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies. Hepatology, 47 (1), pp. 17-24. | Show Abstract | Read more

UNLABELLED: Hepatitis C virus (HCV) infection of Huh-7.5 hepatoma cells results in focal areas of infection where transmission is potentiated by cell-cell contact. To define route(s) of transmission, HCV was allowed to infect hepatoma cells in the presence or absence of antibodies that neutralize cell-free virus infectivity. Neutralizing antibodies (nAbs) reduced cell-free virus infectivity by >95% and had minimal effect(s) on the frequency of infected cells in the culture. To assess whether cell-cell transfer of viral infectivity occurs, HCV-infected cells were cocultured with fluorescently labeled naïve cells in the presence or absence of nAbs. Enumeration by flow cytometry demonstrated cell-cell transfer of infectivity in the presence or absence of nAbs and immunoglobulins from HCV(+) patients. The host cell molecule CD81 and the tight junction protein Claudin 1 (CLDN1) are critical factors defining HCV entry. Soluble CD81 and anti-CD81 abrogated cell-free infection of Huh-7.5 and partially inhibited cell-cell transfer of infection. CD81-negative HepG2 hepatoma cells were resistant to cell-free virus infection but became infected after coculturing with JFH-infected cells in the presence of nAb, confirming that CD81-independent routes of cell-cell transmission exist. Further experiments with 293T and 293T-CLDN1 targets suggested that cell-cell transmission is dependent on CLDN1 expression. CONCLUSION: These data suggest that HCV can transmit in vitro by at least two routes, cell-free virus infection and direct transfer between cells, with the latter offering a novel route for evading nAbs.

Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wölk B, Hatziioannou T, McKeating JA, Bieniasz PD, Rice CM. 2007. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature, 446 (7137), pp. 801-805. | Show Abstract | Read more

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. A better understanding of the viral life cycle, including the mechanisms of entry into host cells, is needed to identify novel therapeutic targets. Although HCV entry requires the CD81 co-receptor, and other host molecules have been implicated, at least one factor critical to this process remains unknown (reviewed in refs 1-3). Using an iterative expression cloning approach we identified claudin-1 (CLDN1), a tight junction component that is highly expressed in the liver, as essential for HCV entry. CLDN1 is required for HCV infection of human hepatoma cell lines and is the first factor to confer susceptibility to HCV when ectopically expressed in non-hepatic cells. Discrete residues within the first extracellular loop (EL1) of CLDN1, but not protein interaction motifs in intracellular domains, are critical for HCV entry. Moreover, antibodies directed against an epitope inserted in the CLDN1 EL1 block HCV infection. The kinetics of this inhibition indicate that CLDN1 acts late in the entry process, after virus binding and interaction with the HCV co-receptor CD81. With CLDN1 we have identified a novel key factor for HCV entry and a new target for antiviral drug development.

von Hahn T, Yoon JC, Alter H, Rice CM, Rehermann B, Balfe P, McKeating JA. 2007. Hepatitis C virus continuously escapes from neutralizing antibody and T-cell responses during chronic infection in vivo. Gastroenterology, 132 (2), pp. 667-678. | Show Abstract | Read more

BACKGROUND & AIMS: Broadly reactive neutralizing antibodies (nAbs) and multispecific T-cell responses are generated during chronic hepatitis C virus (HCV) infection and yet fail to clear the virus. This study investigated the development of autologous nAb and HCV-glycoprotein-specific T-cell responses and their effects on viral sequence evolution during chronic infection in order to understand the reasons for their lack of effectiveness. METHODS: Numerous E1E2 sequences were amplified and sequenced from serum samples collected over a 26-year period from patient H, a uniquely well-characterized, chronically infected individual. HCV pseudoparticles (HCVpp) expressing the patient-derived glycoproteins were generated and tested for their sensitivity to neutralization by autologous and heterologous serum antibodies. RESULTS: A strain-specific nAb response developed early in infection (8 weeks postinfection), whereas cross-reactive antibodies able to neutralize HCVpp-bearing heterologous glycoproteins developed late in infection (>33 wk postinfection). The humoral response continuously failed to neutralize viruses bearing autologous glycoprotein sequences that were present in the serum at a given time. The amplified glycoprotein sequences displayed high variability, particularly in regions corresponding to defined linear B-cell epitopes. Mutations in defined neutralizing epitopes were associated with a loss of recognition by monoclonal antibodies against these epitopes and with decreased neutralization of corresponding HCVpp. Viral escape from CD4 and CD8 T-cell responses also was shown for several novel epitopes throughout the glycoprotein region. CONCLUSIONS: During chronic infection HCV is subjected to selection pressures from both humoral and cellular immunity, resulting in the continuous generation of escape variants.

Blight KJ, McKeating JA, Rice CM. 2002. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol, 76 (24), pp. 13001-13014. | Show Abstract | Read more

Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-alpha) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.

Moore JP, McKeating JA, Weiss RA, Sattentau QJ. 1990. Dissociation of gp120 from HIV-1 virions induced by soluble CD4. Science, 250 (4984), pp. 1139-1142. | Show Abstract | Read more

The CD4 antigen is the high affinity cellular receptor for the human immunodeficiency virus type-1 (HIV-1). Binding of recombinant soluble CD4 (sCD4) or the purified V1 domain of sCD4 to the surface glycoprotein gp120 on virions resulted in rapid dissociation of gp120 from its complex with the transmembrane glycoprotein gp41. This may represent the initial stage in virus-cell and cell-cell fusion. Shedding of gp120 from virions induced by sCD4 may also contribute to the mechanism by which these soluble receptor molecules neutralize HIV-1.

McKeating JA, Griffiths PD, Weiss RA. 1990. HIV susceptibility conferred to human fibroblasts by cytomegalovirus-induced Fc receptor. Nature, 343 (6259), pp. 659-661. | Show Abstract | Read more

The main receptor for the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) on T and B lymphocytes, monocytes and macrophages is the CD4 antigen 1-3. Infection of these cells is blocked by monoclonal antibodies to CD4(1,2) and by recombinant soluble CD4(4-9). Expression of transfected CD4 on the surface of HeLa and other human cells renders them susceptible to HIV infection 10. HIV-antibody complexes can also infect monocytes and macrophages by means of receptors for the Fc portion of immunoglobulins (FcR)11-13), or complement receptors 14,15. The expression of IgG FcRs can be induced in cells infected with human herpes viruses such as herpes simplex virus type 1 (HSV-1)16,17 and human cytomegalovirus (CMV)18-21. Here we demonstrate that FcRs induced by CMV allow immune complexes of HIV to infect fibroblasts otherwise not permissive to HIV infection. Infection was inhibited by prior incubation with human IgG, but not by anti-CD4 antibody or by recombinant soluble CD4. Once HIV had entered CMV-infected cells by means of the FcR, its replication could be enhanced by CMV transactivating factors. Synergism between HIV and herpes viruses could also operate in vivo, enhancing immunosuppression and permitting the spread of HIV to cells not expressing CD4.

Carbonic anhydrase IX in hepatitis B virus associated hepatocellular carcinoma: a bio-marker for predicting disease outcome and therapeutic target.

Background: Hepatocellular carcinoma (HCC) develops from the inflammation-associated injury associated with hepatitis B or C virus infection, obesity, alcohol-related and non-alcoholic steatohepatitis. HCC is the second most common cause for cancer related deaths in the world, with an estimated 750,000 new cases and 695,000 deaths per year. Therapies show limited efficacy and current research efforts focus on the molecular profiling of tumours to identify markers or gene signatures that predict ...

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Understanding the impact of hypoxia on HIV replication and latency

Background:   The lentivirus human immunodeficiency virus type 1 (HIV-1) is not effectively contained by the host immune response, and although currently-available anti-retroviral agents control viral replication and can prevent progression to AIDS they fail to eradicate integrated copies of the viral genome that provide a long-lived cellular reservoir of infection. This necessitates lifelong treatment which is costly and often poorly adhered to; and even if near-complete suppression of virus ...

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