A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: Implications for filovirus budding

VP40, the putative matrix protein of both Ebola and Marburg viruses, posses ses a conserved proline-rich motif(PY motif) at its N terminus. We demonstr ate that the VP40 protein can mediate its own release from mammalian cells, and that the PY motif is important for this self-exocytosis (budding) func tion. In addition, we used Western-ligand blotting to demonstrate that the PY motif of VP40 can mediate interactions with specific cellular proteins t hat have type I WW-domains. including the mammalian ubiquitin ligase. Nedd4 . Single point mutations that disrupted the PY motif of VP40 abolished the PY/WW-domain interactions. Significantly, the full-length VP40 protein was shown to interact both physically and functionally with full-length Rsp5, a ubiquitin ligase of yeast and homolog of Nedd4 The VP40 protein was multiu biquitinated by Rsp5 in a PY-dependent manner in an in vitro ubiquitination assay. These data demonstrate that the VP40 protein of Ebola Virus possess es a PY motif that is functionally similar to those described previously fo r Gag and M proteins of specific retroviruses and rhabdoviruses, respective ly. Last, these studies imply that VP40 likely plays an important role in f ilovirus budding, and that budding of retroviruses. rhabdoviruses, and filo viruses may proceed via analogous mechanisms.