title: CryoEM analysis of capsid assembly and structural changes upon interactions with a host restriction factor, TRIM5α.
abstract: After virus fusion with a target cell, the viral core is released into the host cell cytoplasm and undergoes a controlled disassembly process, termed uncoating, before or as reverse transcription takes place. The cellular protein TRIM5α is a host cell restriction factor that blocks HIV-1 infection in rhesus macaque cells by targeting the viral capsid and inducing premature uncoating. The molecular mechanism of the interaction between capsid and TRIM5α remains unclear. Here, we describe an approach that utilizes cryo-electron microscopy (cryoEM) to examine the structural changes exerted on HIV-1 capsid (CA) assembly by TRIM5α binding. The TRIM5α interaction sites on CA assembly were further dissected by combining cryoEM with pair-wise cysteine mutations that crosslink CA either within a CA hexamer or between CA hexamers. Based on the structural information from cryoEM and crosslinking results from in vitro CA assemblies and purified intact HIV-1 cores, we demonstrate that direct binding of TRIM5α CC-SPRY domains to the viral capsid results in disruption and fragmentation of the surface lattice of HIV-1 capsid, specifically at inter-hexamer interfaces. The method described here can be easily adopted to study other important interactions in multi-protein complexes.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Gongpu
authors.initials: G
authors.lastname: Zhao
authors.forename: Peijun
authors.initials: P
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000090103:Antiviral Restriction Factors; D002213:Capsid / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D020285:Cryoelectron Microscopy / Q000379:methods*; D018360:Crystallography, X-Ray; D003545:Cysteine; D006046:Gold / Q000737:chemistry / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D053768:Metal Nanoparticles; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases
chemical_list: D000090103:Antiviral Restriction Factors; D036022:Capsid Proteins; D002352:Carrier Proteins; D000070584:Tripartite Motif Proteins; D006046:Gold; C477346:TRIM5 protein, human; D044767:Ubiquitin-Protein Ligases; D003545:Cysteine