Background: Retrovirus budding is greatly stimulated by the presence of Gag sequences known as “late” or “L” domains. The L domain of HIV-1 maps to a highly conserved Pro-Thr-Ala-Pro (PTAP) sequence in the p6 domain of Gag. We and others recently observed that the p6 PTAP motif interacts with the cellular endosomal sorting protein TSG101. Consistent with a role for TSG101 in virus release, we demonstrated that over-expressing the N-terminal, Gag-binding domain of TSG101 (TSG-5’) suppresses HIV-1 budding by blocking L domain function.

Methods: TSG101 was subjected to site-directed mutagenesis to map the region that interacts with the HIV-1 p6 L domain. Full-length and mutant TSG101 clones were expressed in HeLa cells to study the effect of mutations on cellular localization, endosomal sorting, and HIV-1 budding.

Results: To elucidate the role of TSG101 in HIV-1 budding, we evaluated the significance of Gag/TSG-5’ binding on the inhibition of HIV-1 release. We observed that a mutation in TSG-5' that disrupts the Gag/TSG101 interaction suppresses the inhibitory activity of TSG-5’. We also determined the effect of over-expressing a panel of truncated TSG101 derivatives and full-length TSG101 (TSG-F) on virus budding. Over-expressing TSG-F inhibits HIV-1 budding; however, the effect of TSG-F on virus release does not require Gag binding. Furthermore, over-expression of the C-terminal portion of TSG101 (TSG-3’) potently inhibits budding of both HIV-1 and murine leukemia virus. Confocal microscopy data indicate that TSG-F and TSG-3’ over-expression induces an aberrant endosome phenotype; this defect is dependent upon the C-terminal, Vps-28-binding domain of TSG101. The effect of over-expressing these various forms of TSG101 on the cellular ESCRT-1 complex was evaluated by FPLC.

Conclusions: The data demonstrate that TSG-5' suppresses HIV-1 release by binding PTAP and blocking HIV-1 L domain function, whereas over-expressing TSG-F or TSG-3’ globally inhibits virus release by disrupting endosomal sorting. Our results highlight the importance of TSG101 and the endosomal sorting pathway in virus budding and suggest that TSG101-based inhibitors can be developed that, like TSG-5’, target HIV-1 without disrupting endosomal sorting.