Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction

The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies viru s (RV) play a key role in both assembly and budding of progeny virions. A P PPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for medi ating interactions with specific cellular proteins containing WW domains. T he PY motif and flanking sequences of the M protein of VSV were used as bai t to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple W W domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeas t homolog of Nedd4, was able to interact both physically and functionally w ith full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M p rotein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding proces s of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrea se the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10-to 20-fold lower tha n those measured from untreated control cells, suggesting that free ubiquit in is important for efficient virus budding. Last, release of a VSV PY muta nt was not inhibited in the presence of MG132, signifying that the function al L domain of VSV is required for the inhibitory effect exhibited by MG132 . These data suggest that the cellular ubiquitin-proteasome machinery is in volved in the budding process of VSV and RV.