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CryoEM Analysis of Capsid Assembly and Structural Changes Upon Interactions with a Host Restriction Factor, TRIM5α

  • Gongpu Zhao
  • Peijun Zhang
Part of the Methods in Molecular Biology book series (MIMB, volume 1087)

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.

Key words

CryoEM Uncoating HIV-1 restriction factor TRIM5α HIV-1 Capsid 

Notes

Acknowledgments

The authors would like to thank Dr. Jinwoo Ahn and Danxia Ke for technical support, and Dr. Teresa Brosenitsch for critical reading of the manuscript. This work was supported by GM082251 and GM085043.

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Gongpu Zhao
    • 1
  • Peijun Zhang
    • 1
  1. 1.Department of Structural BiologyUniversity of Pittsburgh School of MedicinePittsburghUSA

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