Structural mechanisms of HIV-1 inhibition by host cell factors using cryoEM

Project Overview

A schemetic view of HIV-1 and host cell interactions

Infections by retroviruses, such as HIV-1, critically depend on the viral capsid. Many host cell defence proteins, including restriction factors Trim5α, TrimCyp and MxB, target the viral capsid at the early stages of infection and potently inhibit virus replication. These restriction factors appear to function through a remarkable capsid pattern sensing ability that specifically recognizes the assembled capsid, but not the individual capsid protein. Using cutting-edage cryoEM technologies, we aim to determine the molecular interactions between the viral capsid and host restriction factors that underpin their capsid pattern-sensing capability and ability to inhibit HIV-1 replication. Specifically, we will combine cryoEM and cryoET with all-atom molecular dynamics simulations to obtain high-resolution structures, together with mutational and functional analysis, as well as correlative light and cryoEM imaging of viral infection process, to reveal the essential mechanism for HIV-1 capsid recognition and inhibition of HIV-1 infection. Information derived from our studies will allow to design more robust therapeutic agents to block HIV-1 replication.

Training Opportunities

We are located in the Division of Structural Biology, Wellcome Trust Centre for Human Genetics, which provides an ideal environment for multidisciplinary and integrative studies. We also have regular access to eBIC and Diamond Light Source for data collection. Individual projects are tailored to particular student interests and cover techniques in molecular, cellular and structural biology. Through the projects, students will be trained in

Theme

Protein Science & Structural Biology and Immunology & Infectious Disease

Admissions

Project reference number: 866

Funding and admissions information

Supervisors

Name Department Institution Country Email
Peijun Zhang Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine GBR peijun@strubi.ox.ac.uk
Jonathan Grimes Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine GBR jonathan@strubi.ox.ac.uk

Himes BA, Zhang P. 2018. emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging. Nat. Methods, 15 (11), pp. 955-961. Read abstract | Read more

Macromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by cryo-electron tomography (cryo-ET) combined with subtomogram alignment and classification, which in exceptional cases achieves subnanometer resolution, yielding insight into structure-function relationships. However, it remains challenging to apply this approach to specimens that exhibit conformational or compositional heterogeneity or are present in low abundance. To address this, we developed emClarity ( https://github.com/bHimes/emClarity/wiki ), a GPU-accelerated image-processing package featuring an iterative tomographic tilt-series refinement algorithm that uses subtomograms as fiducial markers and a 3D-sampling-function-compensated, multi-scale principal component analysis classification method. We demonstrate that our approach offers substantial improvement in the resolution of maps and in the separation of different functional states of macromolecular complexes compared with current state-of-the-art software. Hide abstract

Alvarez FJD, He S, Perilla JR, Jang S, Schulten K, Engelman AN, Scheres SHW, Zhang P. 2017. CryoEM structure of MxB reveals a novel oligomerization interface critical for HIV restriction. Sci Adv, 3 (9), pp. e1701264. Read abstract | Read more

Human dynamin-like, interferon-induced myxovirus resistance 2 (Mx2 or MxB) is a potent HIV-1 inhibitor. Antiviral activity requires both the amino-terminal region of MxB and protein oligomerization, each of which has eluded structural determination due to difficulties in protein preparation. We report that maltose binding protein-fused, full-length wild-type MxB purifies as oligomers and further self-assembles into helical arrays in physiological salt. Guanosine triphosphate (GTP), but not guanosine diphosphate, binding results in array disassembly, whereas subsequent GTP hydrolysis allows its reformation. Using cryo-electron microscopy (cryoEM), we determined the MxB assembly structure at 4.6 Å resolution, representing the first near-atomic resolution structure in the mammalian dynamin superfamily. The structure revealed previously described and novel MxB assembly interfaces. Mutational analyses demonstrated a critical role for one of the novel interfaces in HIV-1 restriction. Hide abstract

Ning J, Zhong Z, Fischer DK, Harris G, Watkins SC, Ambrose Z, Zhang P. 2018. Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid. J. Virol., 92 (13), Read abstract | Read more

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a human protein that binds HIV-1 capsid and mediates nuclear transport and integration targeting of HIV-1 preintegration complexes. Truncation of the protein at its C-terminal nuclear-targeting arginine/serine-rich (RS) domain produces a protein, CPSF6-358, that potently inhibits HIV-1 infection by targeting the capsid and inhibiting nuclear entry. To understand the molecular mechanism behind this restriction, the interaction between CPSF6-358 and HIV-1 capsid was characterized using and assays. Purified CPSF6-358 protein formed oligomers and bound -assembled wild-type (WT) capsid protein (CA) tubes, but not CA tubes containing a mutation in the putative binding site of CPSF6. Intriguingly, binding of CPSF6-358 oligomers to WT CA tubes physically disrupted the tubular assemblies into small fragments. Furthermore, fixed- and live-cell imaging showed that stably expressed CPSF6-358 forms cytoplasmic puncta upon WT HIV-1 infection and leads to capsid permeabilization. These events did not occur when the HIV-1 capsid contained a mutation known to prevent CPSF6 binding, nor did they occur in the presence of a small-molecule inhibitor of capsid binding to CPSF6-358. Together, our biochemical and transmission electron microscopy data and intracellular imaging results provide the first direct evidence for an oligomeric nature of CPSF6-358 and suggest a plausible mechanism for restriction of HIV-1 infection by CPSF6-358. After entry into cells, the HIV-1 capsid, which contains the viral genome, interacts with numerous host cell factors to facilitate crucial events required for replication, including uncoating. One such host cell factor, called CPSF6, is predominantly located in the cell nucleus and interacts with HIV-1 capsid. The interaction between CA and CPSF6 is critical during HIV-1 replication Truncation of CPSF6 leads to its localization to the cell cytoplasm and inhibition of HIV-1 infection. Here, we determined that truncated CPSF6 protein forms large higher-order complexes that bind directly to HIV-1 capsid, leading to its disruption. Truncated CPSF6 expression in cells leads to premature capsid uncoating that is detrimental to HIV-1 infection. Our study provides the first direct evidence for an oligomeric nature of truncated CPSF6 and insights into the highly regulated process of HIV-1 capsid uncoating. Hide abstract

Liu C, Perilla JR, Ning J, Lu M, Hou G, Ramalho R, Himes BA, Zhao G, Bedwell GJ, Byeon IJ, Ahn J, Gronenborn AM, Prevelige PE, Rousso I, Aiken C, Polenova T, Schulten K, Zhang P. 2016. Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site. Nat Commun, 7 pp. 10714. Read abstract | Read more

The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection. Hide abstract

Zhao G, Perilla JR, Yufenyuy EL, Meng X, Chen B, Ning J, Ahn J, Gronenborn AM, Schulten K, Aiken C, Zhang P. 2013. Mature HIV-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics. Nature, 497 (7451), pp. 643-6. Read abstract | Read more

Retroviral capsid proteins are conserved structurally but assemble into different morphologies. The mature human immunodeficiency virus-1 (HIV-1) capsid is best described by a 'fullerene cone' model, in which hexamers of the capsid protein are linked to form a hexagonal surface lattice that is closed by incorporating 12 capsid-protein pentamers. HIV-1 capsid protein contains an amino-terminal domain (NTD) comprising seven α-helices and a β-hairpin, a carboxy-terminal domain (CTD) comprising four α-helices, and a flexible linker with a 310-helix connecting the two structural domains. Structures of the capsid-protein assembly units have been determined by X-ray crystallography; however, structural information regarding the assembled capsid and the contacts between the assembly units is incomplete. Here we report the cryo-electron microscopy structure of a tubular HIV-1 capsid-protein assembly at 8 Å resolution and the three-dimensional structure of a native HIV-1 core by cryo-electron tomography. The structure of the tubular assembly shows, at the three-fold interface, a three-helix bundle with critical hydrophobic interactions. Mutagenesis studies confirm that hydrophobic residues in the centre of the three-helix bundle are crucial for capsid assembly and stability, and for viral infectivity. The cryo-electron-microscopy structures enable modelling by large-scale molecular dynamics simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer-of-hexamer elements as well as for the entire capsid. Incorporation of pentamers results in closer trimer contacts and induces acute surface curvature. The complete atomic HIV-1 capsid model provides a platform for further studies of capsid function and for targeted pharmacological intervention. Hide abstract