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

Significance HIV-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The assembly of virus particles involves numerous host and viral proteins that are potential therapeutic targets. We used high-resolution microscopy techniques to investigate how the virus hijacks cellular proteins to enable the release of virions from an infected cell. We show with high temporal and spatial resolution that components of the host endosomal sorting complex required for transport (ESCRT) machinery are recruited to the neck of the assembling virus to facilitate scission of the link between the virus and the cell. 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.