Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid

Xin Meng,Angela M Gronenborn,Peijun Zhang,Gongpu Zhao,Jiying Ning,Christopher Aiken,Maria Delucia,Jinwoo Ahn,Danxia Ke,Ernest Yufenyuy,John A T Young

doi:10.1371/journal.ppat.1002886

Abstract

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles.

Highlights

The Gag polyprotein encompasses the major structural elements responsible for assembly of retroviruses, including human immunodeficiency virus type 1 (HIV-1)
HIV-1 virions assemble as immature particles that must undergo proteolytic maturation to become infectious
The Gag polyprotein is cleaved into matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins, and CA assembles to form a mature viral capsid

Summary

Introduction

The Gag polyprotein encompasses the major structural elements responsible for assembly of retroviruses, including human immunodeficiency virus type 1 (HIV-1). And budding involve Gag self-association and interactions with the host cell plasma membrane, the viral genomic RNA, and host cell dependency factors, leading to the formation of immature, non-infectious, spherical virus particles [1,2]. The immature particles undergo maturation, resulting in a dramatic morphological rearrangement of viral components [3,4]. This process is initiated by proteolytic cleavage of Gag into the structural proteins, matrix (MA), capsid (CA), and nucleocapsid (NC), as well as additional peptide sequences that vary among retroviruses (p6 and spacer peptides, SP1 and SP2, in the case of HIV-1) [5]. Several models have been proposed, including de novo assembly of CA monomers or hexamers after proteolysis [22] and trigger-mediated conformational switch mechanisms [36,37]

Author Summary

Materials and Methods