The long-term objective of this proposal is to elucidate mechanisms in late retrovirus replication that involve budding of virus from cells. Experiments are proposed to follow up our co-discovery of the retrovirus L domain required for budding of virus from cells and our subsequent identification of two potential cellular proteins (Nedd4 and TSG1O1), both involved with ubiquitination, that interact with the RSV and HIV-1 L domains, respectively. Experiments are proposed to prove that these cell proteins are the biological partners required for virus budding. We will look for co-localization of the cellular proteins with Gag inside cells, disruption of virus budding in cells by dominant negative expression of fragments of the cell proteins, and coimmune precipitation of Gag with the respective cell proteins from extracts that is dependent upon the L domain. Having established in vivo evidence that the two cell proteins interact with the respective L domain sequences of Gag, cells lines containing genetic or phenotypic knockouts of the candidate cellular proteins will be constructed and used to examine their roles in the budding process. In addition, immune precipitation techniques using Gag constructs with and without L domain sequences will be used to recover additional cellular proteins that may be involved in the budding process. The identity of these proteins will be established using immune and biochemical techniques and their role in budding will be established as described above. This will begin to define a pathway of interactions required to bud virus from cells. In a separate direction, we will carry out a novel genetic analysis of the sequence requirements for interaction of the L domains with their cell partners by a novel in vivo mutagenesis procedure. These studies will set the groundwork for looking at the budding mechanism in detail. Moreover, the finding that the RSV L domain PY motif is found in a broad class of enveloped viruses (including rhabdo-, filo-, and herpesviruses) has wide ranging implications for the development of antiviral agents directed at cell proteins involved in release of viruses from cells.