| HIV-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 e&barbelow;ndosomal s&barbelow;orting c&barbelow;omplexes r&barbelow;equired for t&barbelow;ransport (ESCRT) pathway and are required for inward vesicle formation at late endosomes or m&barbelow;ultiv&barbelow;esicular b&barbelow;odies (MVBs). Most ESCRT proteins are subunits one of five hetero-oligomeric complexes named ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III, and the VPS4 AAA ATPases (v&barbelow;acuolar p&barbelow;rotein s&barbelow;orting), 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 c&barbelow;harged m&barbelow;ultivesicular body p&barbelow;roteins (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.;Chapter 2 describes my contribution to the identification of the protein network involved in HIV-1 budding. Discovery of new protein-protein interactions in the ESCRT pathway defined bridges between complexes that act early in the pathway and those that act late. Chapter 3 describes the role of ESCRT-I in HIV-1 budding. Specifically, I identified four previously unknown subunits of human ESCRT-I (VPS37A-D) and characterized the biochemistry of VPS37B. Finally, Chapter 4, Appendix and Appendix B describe the interaction between the CHMPs of ESCRT-III and the VPS4 MIT domains. My contribution to these sections was the identification of the regions of required for the complex interactions. Specifically, I identified a C-terminal motif found in CHMP 1-3 that binds the VPS4 MIT domains. Structural studies of the VPS4B/CHMPB complex revealed that the CHMP2B peptide binds to the antiparallel three helix bundle of the VPS4B MIT domain between helices two and three, and suggested two plausible models for the complex. Overall, the data presented here support the model that HIV-1 usurps the human ESCRT pathway to enable virus budding, and help to define the interactions that allow this pathway to facilitate MVB vesicle formation and HIV-1 budding. |
