HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and −2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants.

Tsg101, a component of the endosomal sorting complex required for transport (ESCRT), is responsible for recognition of events requiring the machinery, as signaled by cargo tagging with ubiquitin (Ub), and for recruitment of downstream acting subunits to the site. Although much is known about the latter function, little is known about its role in the earlier event. The N-terminal domain of Tsg101 is a structural homologue of Ub conjugases (E2 enzymes) and the protein associates with Ub ligases (E3 enzymes) that regulate several cellular processes including virus budding. A pocket in the domain recognizes a motif, PT/SAP, that permits its recruitment. PT/SAP disruption makes budding dependent on Nedd4L E3 ligases. Using HIV-1 encoding a PT/SAP mutation that makes budding Nedd4L-dependent, we identified as critical for rescue the residues in the catalytic (HECT) domain of the E3 enzyme that lie in proximity to sites in Tsg101 that bind Ub non-covalently. Mutation of these residues impaired rescue by Nedd4L but the same mutations had no apparent effect in the context of a Nedd4 isomer, Nedd4-2s, whose N-terminal (C2) domain is naturally truncated, precluding C2-HECT auto-inhibition. Surprisingly, like small molecules that disrupt Tsg101 Ub-binding, small molecules that interfered with Nedd4 substrate recognition arrested budding at an early stage, supporting the conclusion that Tsg101–Ub–Nedd4 interaction promotes enzyme activation and regulates Nedd4 signaling for viral egress. Tsg101 regulation of E3 ligases may underlie its broad ability to function as an effector in various cellular activities, including viral particle assembly and budding.