Dynamics of HIV-1 Assembly and Release

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.

Properties and functions of the nucleocapsid protein in virus assembly

“…141,142 Once Gag-NC-genomic RNA complexes start to be formed, when and how are they targeted to the site of virus assembly (at the plasma membrane or other cellular membranes) is poorly understood. The recent advances in the visualization of Gag trafficking and virus assembly in live cells 46,[143][144][145][146][147][148] should allow in the future the discovery of the sequential events that guide particle assembly. The challenge will be to define which cellular factors act during the pathway from Gag synthesis to particle assembly, and where and how they act in this pathway.…”

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

“…Once these complexes become anchored in the plasma membrane, Pr55 Gag molecules are rapidly recruited as viral particle assembly proceeds 43,46 .…”

Quantitative Super-Resolution Imaging Reveals Protein Stoichiometry and Nanoscale Morphology of Assembling HIV-Gag Virions

“…Viral assembly is widely studied using HIV-Gag, the main structural protein of HIV, which is sufficient to drive the assembly of virus-like particles (VLPs) in the absence of other viral components 1,2 . Fluorescence imaging has been used to follow the time-dependent increase in the intensity of Gag clusters in living cells 3,4 , revealing the time scale of virion formation. Electron microscopy (EM) has elucidated the spatial arrangement of Gag in fully formed virions [5][6][7][8] .…”