Doctor of Philosophy

dissertationHIV-1 is an enveloped RNA virus that recruits cellular machinery to facilitate virus budding. HIV-1 Gag, the structural protein that drives the assembly and budding processes, binds directly to two cellular factors, TSG101 and ALIX. Both TSG101 and ALIX are proteins in the endosomal sorting complexes required for transport (ESCRT) pathway and are required for inward vesicle formation at late endosomes or multivesicular bodies (MVBs). Most ESCRT proteins are subunits one of five heterooligomeric complexes named ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III, and the VPS4 AAA ATPases (vacuolar protein sorting), which are sequentially recruited to sites of vesicle formation and virus budding. TSG101 is a member of the ESCRT-I complex and is involved in recognition of cargos and recruitment of ESCRT-II. ESCRT-II recruits ESCRT-III, which is composed of charged multivesicular body proteins (CHMPs) that coassemble to form a lattice on the surface of endosomal membranes. ALIX interacts with proteins in both ESCRT-I and ESCRT-III, but is not a constitutive member of either complex. ESCRT-III proteins bind directly to the VPS4 AAA ATPases, which are the only known enzymes in the pathway. ATP hydrolysis by the VPS4 ATPases releases all assembled ESCRT machinery, allowing multiple rounds of vesicle formation or viral egress. This thesis focuses on the identification and characterization of components of the human ESCRT protein network involved in HIV-1 budding