Live-cell visualization of dynamics of HIV budding site interactions with an ESCRT component

Abstract

HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation. Recruitment of ESCRT by late-domain motifs in the viral Gag polyprotein and a role of ESCRT in HIV release are firmly established, but the order of events, their kinetics and the mechanism of action of individual ESCRT components in HIV budding are unclear at present. Using live-cell imaging, we show late-domain-dependent recruitment of VPS4A to nascent HIV particles at the host cell plasma membrane. Recruitment of VPS4A was transient, resulting in a single or a few bursts of at least two to five VPS4 dodecamers assembling at HIV budding sites. Bursts lasted for ∼35 s and appeared with variable delay before particle release. These results indicate that VPS4A has a direct role in membrane scission leading to HIV-1 release.

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

“…This final step is thought to be mediated by ESCRT-III, possibly in conjuction with the ATPase VPS4 (33,49). In the conception of these experiments, we had anticipated that inefficient release of Gag puncta into the GUV interior might be seen even in the absence of ESCRTs, because high levels of Gag overexpression have been shown to drive budding (50).…”

Antiviral drug discovery: broad-spectrum drugs from nature

“…siRNA downregulation of components of the ESCRT and associated factors such as ALIX has been shown to block cell entry of VSV, LFV, and LCMV, and cell exit of HIV and hepatitis A virus (HAV) 56,58,61,62,87 . However, a non-toxic chemical compound has not yet been released for clinical use.…”

“…However, as viruses are bound to utilize the host cellular machinery to propagate, they are critically dependent on cellular factors that are up-or downregulated as needed. Examples are the down-regulation of membrane receptors [48][49][50][51][52][53] , the up-regulation of the lipid metabolism 54 , the use of the mRNA processing machinery 55 or the hijacking of components of the endosomal-sorting complex (ESCRT) required for virus export from infected cells [56][57][58][59][60][61][62][63] (see 2.4 for details). Moreover, as the life cycle of different viruses share common cellular factors and pathways, it is feasible that these could be used as targets for the design of broad-spectrum antivirals.…”

Vfa1 Binds to the N-terminal Microtubule-interacting and Trafficking (MIT) Domain of Vps4 and Stimulates Its ATPase Activity

“…Finally, the ATPase Vps4 is recruited to the site of vesicle formation. Through ATP hydrolysis, Vps4 is likely to drive the completion of vesicle formation in an irreversible direction and ultimately provide energy for the entire process (17,18). Biochemically, Vps4 catalyzes the removal of ESCRT-III oligomers from the endosomal membrane (19).…”