Dynamics of ESCRT protein recruitment during retroviral assembly

The ESCRT (Endosomal Sorting Complex Required for Transport) complexes and associated proteins mediate membrane scission reactions, such as multi-vesicular body formation, the terminal stages of cytokinesis and retroviral particle release. These proteins are believed to be sequentially recruited to the site of membrane scission, and then complexes are disassembled by the ATPase Vps4A. However these events have never been observed in living cells and their dynamics are unknown. By quantifying the recruitment of several ESCRT and associated proteins during the assembly of two retroviruses, we show that Alix progressively accumulated at viral assembly sites, coincident with the accumulation of the major viral structural protein, Gag, and was not recycled after assembly. In contrast, ESCRT-III and Vps4A were only transiently recruited when the accumulation of Gag was complete. These data suggest that the rapid and transient recruitment of proteins that act late in the ESCRT pathway and carry out membrane fission is triggered by prior and progressive accumulation of proteins that bridge viral proteins and the late-acting ESCRT proteins.