Proteins related to the Nedd4 family of ubiquitin protein ligases interactwith the L domain of Rous sarcoma virus and are required for gag budding from cells

The late assembly (L) domain of retrovirus Gag, required in the final steps of budding for efficient exit from the host cell, is thought to mediate it s function through interaction with unknown cellular factors. Here, we repo rt the identification of the Nedd4-like family of E3 ubiquitin protein liga ses as proteins that specifically interact with the Rous sarcoma virus (RSV ) L domain in vitro and in vivo. We screened a chicken embryo cDNA expressi on library by using a peptide derived from the RSV p2b sequence, isolating two unique partial cDNA clones. Neither clone interacted with a peptide con taining mutations known to disrupt in vivo RSV L domain function or with hu man immunodeficiency virus type 1 (HIV-1) and equine infectious anemia viru s (EIAV) L domain-derived peptides. The WW domain region of one of the clon es, late domain-interacting protein 1 (LDI-1), but not the C2 domain, bound RSV Gag and inhibited RSV Gag budding from human 293 cells in a dominant-n egative manner, functionally implicating LDI-1 in RSV particle budding from cells. RSV Gag can be coimmune precipitated from cell extracts with an ant isera directed at an exogenously expressed hemagglutinin (HA)-tagged LDI-1 or endogenous Nedd4 proteins. These findings mechanistically link the cellu lar ubiquitination pathway to retrovirus budding.