In vitro analysis of human immunodeficiency virus particle dissociation: gag proteolytic processing influences dissociation kinetics.

Barbara Müller,Jochen Reinstein,Maria Anders,Fabrizio Mammano

doi:10.1371/journal.pone.0099504

Abstract

Human immunodeficiency virus particles undergo a step of proteolytic maturation, in which the main structural polyprotein Gag is cleaved into its mature subunits matrix (MA), capsid (CA), nucleocapsid (NC) and p6. Gag proteolytic processing is accompanied by a dramatic structural rearrangement within the virion, which is necessary for virus infectivity and has been proposed to proceed through a sequence of dissociation and reformation of the capsid lattice. Morphological maturation appears to be tightly regulated, with sequential cleavage events and two small spacer peptides within Gag playing important roles by regulating the disassembly of the immature capsid layer and formation of the mature capsid lattice. In order to measure the influence of individual Gag domains on lattice stability, we established Förster's resonance energy transfer (FRET) reporter virions and employed rapid kinetic FRET and light scatter measurements. This approach allowed us to measure dissociation properties of HIV-1 particles assembled in eukaryotic cells containing Gag proteins in different states of proteolytic processing. While the complex dissociation behavior of the particles prevented an assignment of kinetic rate constants to individual dissociation steps, our analyses revealed characteristic differences in the dissociation properties of the MA layer dependent on the presence of additional domains. The most striking effect observed here was a pronounced stabilization of the MA-CA layer mediated by the presence of the 14 amino acid long spacer peptide SP1 at the CA C-terminus, underlining the crucial role of this peptide for the resolution of the immature particle architecture.

Highlights

And release of human immunodeficiency virus (HIV1) progeny are orchestrated by the viral structural protein Gag [1,2], which assembles at the plasma membrane of virus producing cells and recruits the viral RNA genome as well as other virion proteins and host cell factors to the viral budding site
Proteolytic processing of Gag leads to a dramatic structural rearrangement of its subunits within the virion, with MA lining the lipid envelope of the mature particle and NC complexing and condensing the viral genome within a conical capsid assembled from CA subunits
We have established a Forster’s resonance energy transfer (FRET) based in vitro assay (Figure 1A) to monitor dissociation of mature, immature and partially processed HIV-1 virions released from particle producing cells with high time resolution and employed this system to compare dissociation kinetics of particles consisting of Gag proteins comprising different domains of the polyprotein precursor

Summary

Introduction

And release of human immunodeficiency virus (HIV1) progeny are orchestrated by the viral structural protein Gag [1,2], which assembles at the plasma membrane of virus producing cells and recruits the viral RNA genome as well as other virion proteins and host cell factors to the viral budding site (reviewed in [2]). We have established a FRET based in vitro assay (Figure 1A) to monitor dissociation of mature, immature and partially processed HIV-1 virions released from particle producing cells with high time resolution and employed this system to compare dissociation kinetics of particles consisting of Gag proteins comprising different domains of the polyprotein precursor.

Results

Conclusion