Dynamics of HIV-1 Assembly and Release

Sergey Ivanchenko,Hans-Georg Kräusslich,Karl Rohr,Marko Lampe,Don C Lamb,Christoph Bräuchle,Roland Eils,William J Godinez,Barbara Müller,Walther Mothes

doi:10.1371/journal.ppat.1000652

Abstract

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10−3 s−1, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.

Highlights

And release of progeny virions are fundamental steps in viral replication
Results from pulsechase labeling, density gradient fractionation, time-lapse fluorescence imaging and intracellular fluorescence resonance energy transfer measurements have resulted in a current general view of retroviral assembly: Gag is rapidly converted from a soluble form to a multimeric, membrane associated complex and this process is reflected by changes in the distribution of Gag within the cell (e.g. [5,6,7,8,9,10])
To determine whether Gag molecules arrive at the assembly site directly from the cytosol or by lateral diffusion within the plasma membrane, we made use of an human immunodeficiency virus (HIV)-1 variant labeled with a thermostable mutant of the photoconvertible protein mEosFP

Summary

Introduction

In the case of retroviruses, such as human immunodeficiency virus type 1 (HIV-1), the viral structural polyprotein Gag plays a central role in mediating both of these processes which occur concomitantly at the plasma membrane of the infected cell. Gag comprises domains required for membrane binding, multimerization, nucleic acid binding as well as for interaction with the host cell derived budding machinery and has been demonstrated to direct the formation of virus like particles (VLP) in the absence of other viral proteins [1]. The assembly process, leading from monomeric Gag molecules translated at cytoplasmic polysomes to the virus bud comprising several thousand Gag molecules at the plasma membrane, has been investigated using a variety of techniques. Oligo- or multimers of Gag have been detected in the cytoplasm and at intracellular membranes [10,19] and are believed to be assembly intermediates, but the size of Gag

Author Summary

Conclusions

Findings

Materials and Methods