||The cellular protein ALIX and other ESCRT proteins facilitate topologically equivalent membrane abscission events, including viral envelope separation from host membranes, biogenesis of multi-vesicular bodies, and midbody scission at the late stage of cytokinesis. Late domain motifs displayed by retroviral Gag polyproteins are responsible for recruiting ESCRT proteins. The three best-characterized classes of late domains are: the "P(S/T)AP" late domains that bind TSG101 of the ESCRT-I complex, the "PPXY" late domains that bind NEDD4 family ubiquitin E3 ligases, and the "YPXnL" late domains that bind ALIX. ALIX also binds the ESCRT-III protein CHMP4, which recruits other ESCRT-III subunits and VPS4 complexes to carry out membrane fission. My work in this dissertation is centered on how ALIX is recruited by various retroviruses and how ALIX function is regulated in viral budding. We first determined crystal structures of ALIXBro1, ALIXV and ALIXBro1-V. Second, in order to understand how the viral Gag proteins hijack ALIX, we determined the structure of ALIXBro1-V in complex with HIV and EIAV YPXnL late motifs. Third, we used surface plasmon resonance (SPR) to map a new type of ALIX-binding elements from certain SIV strains, which do not contain the canonical YPXnL late domains and still package ALIX in the virions. Furthermore, the new ALIX-binding motifs were crystallized with ALIXBro1-V. All these late-domain ligands adopt different conformations of backbones to interact with the equivalent interface on the ALIX V domain. Based on sequence analysis, nearly every known primate lentiviruses contains an ALIX-binding site, suggesting that the ability to recruit ALIX provide a strong selective advantage for viruses. Fourth, we discovered that the fulllength ALIX is autoinhibited by its C-terminal proline-rich region (PRR), which blocks the interaction of viral late domains based on the results of isothermal titration calorimetry (ITC), SPR and small-angle X-ray scattering (SAXS). The mutation that opens the closed conformation of the V domain partitioned ALIX into membrane-containing fractions and enhanced virus budding. These observations suggest that the function of ALIX is highly regulated, and ALIX activation requires dissociation of the autoinhibitory PRR, opening of the V domain, and probably protein dimerization.