Analysis of the Initiating Events in HIV-1 Particle Assembly and Genome Packaging

Sebla B Kutluay,Paul D Bieniasz,Thomas J Hope

doi:10.1371/journal.ppat.1001200

Sebla B Kutluay, Paul D Bieniasz + Show 1 more

Open Access

https://doi.org/10.1371/journal.ppat.1001200

Copy DOI

Abstract

HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells.

Highlights

Of human immunodeficiency virus type 1 (HIV-1) is a multi-step process that is driven and coordinated by the viral Gag protein
Gag only becomes visible at the site of the membrane-anchored RNA at later times, suggesting that a small number of Gag molecules are initially responsible for retaining viral RNA at the plasma membrane [43]
Because the initial interaction between Gag and RNA apparently involves an invisible number of Gag molecules, visual techniques are limited in their ability to analyze earlier assembly events, i.e. whether the initial Gag-RNA interaction occurs in the cytoplasm, or at the plasma membrane

Summary

Introduction

Of human immunodeficiency virus type 1 (HIV-1) is a multi-step process that is driven and coordinated by the viral Gag protein. The Gag:RNA interaction is mediated by the nucleocapsid (NC) domain of Gag that binds directly to the packaging sequence (psi), which is composed of four stem loops located within the 59 UTR and the 59 end of the gag gene [1,2,3,4]. Another key event is the recruitment of Gag molecules to the plasma membrane, the major site for productive HIV-1 assembly [5,6,7]. After the release of particles from the cell surface, a number of proteolytic cleavage events in Gag lead to major structural changes, yielding infectious virions

Objectives

Methods

Results

Conclusion