title: Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding.
abstract: HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the endosomal sorting complex required for transport (ESCRT) machinery to enable particle release. However, little is known about the temporal and spatial organization of ESCRT protein recruitment. Using multiple-color live-cell total internal reflection fluorescence microscopy, we observed that the ESCRT-I protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly, whereas later-acting ESCRT proteins (Chmp4b and Vps4A) are recruited sequentially, once Gag assembly is completed. Chmp4b, a protein that is required to mediate particle scission, is recruited to HIV-1 assembly sites ∼10 s before the ATPase Vps4A. Using two-color superresolution imaging, we observed that the ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the periphery of the nascent virions, with the Tsg101 assemblages positioned closer to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are consistent with the notion that the ESCRT machinery is recruited transiently to the neck of the assembling particle and is thus present at the appropriate time and place to mediate fission between the nascent virus and the plasma membrane.
authors.affiliation: Laboratory of Cellular Biophysics and.
authors.forename: Marina
authors.initials: M
authors.lastname: Bleck
authors.affiliation: Laboratory of Cellular Biophysics and.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Itano
authors.affiliation: Laboratory of Cellular Biophysics and.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Johnson
authors.affiliation: Bio-Imaging Resource Center, The Rockefeller University, New York, NY 10065; and.
authors.forename: V Kaye
authors.initials: VK
authors.lastname: Thomas
authors.affiliation: Bio-Imaging Resource Center, The Rockefeller University, New York, NY 10065; and.
authors.forename: Alison J
authors.initials: AJ
authors.lastname: North
authors.affiliation: Howard Hughes Medical Institute, Aaron Diamond AIDS Research Center, Laboratory of Retrovirology, The Rockefeller University, New York, NY 10016.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Bieniasz
authors.affiliation: Laboratory of Cellular Biophysics and simon@rockefeller.edu.
authors.forename: Sanford M
authors.initials: SM
authors.lastname: Simon
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism; D019065:Virus Assembly; D057074:Virus Release
chemical_list: C478397:CHMP4B protein, human; D056827:Endosomal Sorting Complexes Required for Transport; D025262:Vacuolar Proton-Translocating ATPases; D000074183:ATPases Associated with Diverse Cellular Activities; C490305:VPS4A protein, human
keywords: TIR-FM; nanobody; single molecule localization microscopy; viral assembly
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