CryoET structures of immature HIV Gag reveal six-helix bundle

Luiza Mendonça,Xiaofeng Fu,Jiwei Liu,Eric O Freed,Christopher Aiken,Thomas Frosio,Peijun Zhang,Benjamin A Himes,Jiying Ning,Mateusz Olek,Dapeng Sun,Abhay Kotecha,Jing Zhou,Alex B Kleinpeter

doi:10.1038/s42003-021-01999-1

Abstract

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.

Highlights

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses
spacer peptide 1 (SP1) T8I is a compensatory mutation that emerged in maturation inhibitors (MIs)-dependent viruses subject to the selective pressure of replicating under sub-optimal concentrations of MIs
We used two different Gag assemblies for structure analysis by cryo-electron tomography (cryoET): GagΔMAT8I protein assemblies expressed in E. coli (Fig. 1a and b) and GagT8I viral-like particles (VLPs) produced in HEK293T cells (Fig. 1a and d)

Summary

Introduction

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and block HIV-1 infection. The structures of immature CA lattice and the first 8 residues of SP1 were determined previously by cryoEM and X-ray crystallography[11,12] Both structures show a six-helix bundle formed by a stretch of 16 residues, 8 from the CA C-terminal domain (CACTD) and 8 from SP1. The rest of SP1 and its connection to NC could not be resolved in these structures

Methods

Results

Conclusion