Structural and functional studies of ALIX/AIP1 in HIV-1 budding

Budding is an important step in the life-cycle of enveloped viruses such as HIV-1, where viral particles bud through the plasma or endosomal membranes to acquire their lipid envelope. Budding occurs late in the viral life-cycle and is the final step before a virus undergoes proteolytic maturation to produce an infectious virion. Viral budding requires a number of host cellular factors that facilitate the budding process and appear to be important for membrane curvature generation/stabilization or membrane fission events. Two such cellular factors that are involved in budding are TSG101 and ALIX, which are recruited to sites of budding through direct interactions with the viral Gag protein, and function to recruit additional budding machinery. Both TSG101 and ALIX normally function in vesicle budding at the late endosome during multivesicular body (MVB) biogenesis, where cargo molecules such as cell surface receptors are sorted into vesicles of the MVB for subsequent degradation in the lysosome. Both viral and vesicle budding undergo topologically similar events and the current hypothesis is that viruses have usurped cellular machinery normally functioning at the MVB to facilitate viral budding. In order to gain insight into this process, my work in this thesis has focused on structural and functional studies of ALIX with the goal of understanding how ALIX recognizes viruses and recruits additional cellular machinery to sites of budding. To this end, I have determined the crystal structure of a construct spanning the structured regions of ALIX and shown that the protein is composed of uniquely structured Bro1 and V domains that are providing insight into the mechanism of ALIX's function. Furthermore, I have collaborated to determine the structure of ALIX in complex with the viral peptide recognition sequence as well as the structure of ALIX in complex with CHMP4 proteins, components of the cellular machinery required for viral and vesicle budding. The structural and biochemical characterization of these interactions in this thesis has provided a basis for understanding how ALIX acts as a scaffold to link viral proteins or cellular cargo molecules to the viral and vesicle budding pathways.