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.