title: 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 approximately 5 x 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 approximately 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.
authors.affiliation: Physical Chemistry, Department of Chemistry and Biochemistry, Munich Center for Integrated Protein Science (CiPSM) and Center for NanoScience, Ludwig-Maximilians-Universität München, Munich, Germany.
authors.forename: Sergey
authors.initials: S
authors.lastname: Ivanchenko
authors.forename: William J
authors.initials: WJ
authors.lastname: Godinez
authors.forename: Marko
authors.initials: M
authors.lastname: Lampe
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Kräusslich
authors.forename: Roland
authors.initials: R
authors.lastname: Eils
authors.forename: Karl
authors.initials: K
authors.lastname: Rohr
authors.forename: Christoph
authors.initials: C
authors.lastname: Bräuchle
authors.forename: Barbara
authors.initials: B
authors.lastname: Müller
authors.forename: Don C
authors.initials: DC
authors.lastname: Lamb
mesh_terms: D002460:Cell Line; D002462:Cell Membrane; D015683:Gene Products, gag / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D015336:Molecular Probe Techniques; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly
chemical_list: D015683:Gene Products, gag