ALIX Is a Lys63-Specific Polyubiquitin Binding Protein that Functions in Retrovirus Budding

The diversity of ubiquitin (Ub)-dependent signaling is attributed to the ability of this small protein to form different types of covalently linked polyUb chains and to the existence of Ub binding proteins that interpret this molecular syntax. We used affinity capture/mass spectrometry to identify ALIX, a component of the ESCRT pathway, as a Ub binding protein. We report that the V domain of ALIX binds directly and selectively to K63-linked polyUb chains, exhibiting a strong preference for chains composed of more than three Ub. Sequence analysis identified two potential Ub binding sites on a single α-helical surface within the coiled-coil region of the V domain. Mutation of these putative Ub binding sites inhibited polyUb binding to the isolated V domain in vitro and impaired budding of lentiviruses. These data reveal an important role for K63 polyUb binding by ALIX in retroviral release. ► The ESCRT protein ALIX binds selectively to K63-linked polyubiquitin chains ► K63 Ub chains bind to conserved amino acid triads in the coiled-coil V domain ► PolyUb binding by ALIX contributes to budding of human and equine lentiviruses Dowlatshahi et al. find that the V domain of ALIX, a component of the ESCRT pathway, binds directly and selectively to K63-linked polyubiquitin chains. Mutating ALIX to disrupt this binding also impairs HIV and EIAV lentivirus release and infectivity.