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Proc Natl Acad Sci U S A. 2014 Aug 19;111(33):12211-6. doi: 10.1073/pnas.1321655111. Epub 2014 Aug 6.

Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding.

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Laboratory of Cellular Biophysics and.
Bio-Imaging Resource Center, The Rockefeller University, New York, NY 10065; and.
Howard Hughes Medical Institute, Aaron Diamond AIDS Research Center, Laboratory of Retrovirology, The Rockefeller University, New York, NY 10016.
Laboratory of Cellular Biophysics and


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.


TIR-FM; nanobody; single molecule localization microscopy; viral assembly

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