Replication of RNA and small DNA viruses in mammalian cells requires a genome that codes for viral proteins and an ability to evade sophisticated and powerful interferon-coupled cellular defences and inflammatory and adaptive immune responses in the host. Viruses have developed a series of counter-measures to prevent induction of interferon (IFN)-β on infection of cells and disrupt systemic response mediated through cytokines and IFN-γ.
Conventionally, these viral evasion mechanisms are mediated through the production of a range of virally encoded proteins that serve to interact with and block or degrade components in the cellular response pathway. However, we have discovered that more fundamental aspects of virus structure, such as its genome composition and ability to form RNA structures, also modify cellular interactions and play roles in virus persistence and in optimising replication rates to maximise their evolutionary fitness. For example, we have recently discovered evidence that CpG and UpA dinucleotides in RNA viruses play central roles in inducing host innate and adaptive immune responses on infection. Compositional selection against these dinucleotides in mammalian RNA virus genomes remains unexplained functionally although we believe it is part of a currently uncharacterised self-non-self recognition system coupled to innate immunity. The recent discovery of targeting of high CpG sequences in the HIV-1 genome and preventing gene expression may represent one element in this pathway.
These interactions have profound downstream effects on virus pathogenicity and outcomes of infections. Recently, we constructed influenza A virus (IAV) mutants with maximised frequencies of CpG and UpA dinucleotides in segment 5. They displayed major replication defects in cell culture compared to wild type virus. Attenuation was also manifested in vivo, with 10-100 fold reduced viral loads in lungs of mice infected with 200 PFU of CpG-high and UpA-high mutants. However, both induced powerful inflammatory cytokine and adaptive (T cell and neutralising antibody) responses disproportionate to their replication.
In the DPhil project, we will further develop this model to understand better the role of virus composition in viral pathogenesis and investigate aspects of the relationship between replication ability, pathogenicity and host immune response
The DPhil will provide experience and training in a wide range of laboratory molecular virology methods, in vivo studies and in immunology. These include:
The work is at the forefront of our growing understanding of the remarkably complex regulatory systems underlying gene expression and how these can be manipulated for biotechnology purposes. The project will take place alongside groups of scientist working the supervisors’ groups investigating other aspects of viral pathogenesis and HCV biology / immunology.
Project reference number: 814
|Professor Peter Simmonds||Experimental Medicine Division||Oxford University, Peter Medawar Building||GBRfirstname.lastname@example.org|
|Professor Ervin Fodor||Dunn School of Pathology||University of Oxford||GBRemail@example.com|
Most vertebrate and plant RNA and small DNA viruses suppress genomic CpG and UpA dinucleotide frequencies, apparently mimicking host mRNA composition. Artificially increasing CpG/UpA dinucleotides attenuates viruses through an entirely unknown mechanism. Using the echovirus 7 (E7) model in several cell types, we show that the restriction in E7 replication in mutants with increased CpG/UpA dinucleotides occurred immediately after viral entry, with incoming virions failing to form replication complexes. Sequences of CpG/UpA-high virus stocks showed no evidence of increased mutational errors that would render them replication defective, these viral RNAs were not differentially sequestered in cytoplasmic stress granules nor did they induce a systemic antiviral state. Importantly, restriction was not mediated through effects on translation efficiency since replicons with high CpG/UpA sequences inserted into a non-coding region were similarly replication defective. Host-cells thus possess intrinsic defence pathways that prevent replication of viruses with increased CpG/UpA frequencies independently of codon usage. Hide abstract
Previously, we demonstrated that frequencies of CpG and UpA dinucleotides profoundly influence the replication ability of echovirus 7 (Tulloch et al., 2014). Here, we show that that influenza A virus (IAV) with maximised frequencies of these dinucleotides in segment 5 showed comparable attenuation in cell culture compared to unmodified virus and a permuted control (CDLR). Attenuation was also manifested in vivo, with 10-100 fold reduced viral loads in lungs of mice infected with 200PFU of CpG-high and UpA-high mutants. However, both induced powerful inflammatory cytokine and adaptive (T cell and neutralising antibody) responses disproportionate to their replication. CpG-high infected mice also showed markedly reduced clinical severity, minimal weight loss and reduced immmunopathology in lung, yet sterilising immunity to lethal dose WT challenge was achieved after low dose (20PFU) pre-immunisation with this mutant. Increasing CpG dinucleotide frequencies represents a generic and potentially highly effective method for generating safe, highly immunoreactive vaccines. Hide abstract
Mutating RNA virus genomes to alter codon pair (CP) frequencies and reduce translation efficiency has been advocated as a method to generate safe, attenuated virus vaccines. However, selection for disfavoured CPs leads to unintended increases in CpG and UpA dinucleotide frequencies that also attenuate replication. We designed and phenotypically characterised mutants of the picornavirus, echovirus 7, in which these parameters were independently varied to determine which most influenced virus replication. CpG and UpA dinucleotide frequencies primarily influenced virus replication ability while no fitness differences were observed between mutants with different CP usage where dinucleotide frequencies were kept constant. Contrastingly, translation efficiency was unaffected by either CP usage or dinucleotide frequencies. This mechanistic insight is critical for future rational design of live virus vaccines and their safety evaluation; attenuation is mediated through enhanced innate immune responses to viruses with elevated CpG/UpA dinucleotide frequencies rather the viruses themselves being intrinsically defective. Hide abstract
Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1-1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-β, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host-pathogen interactions. Hide abstract