Role of HIV-1 p6gag and Ubiquitin in Virus Release

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Retroviral Gag polyproteins have specific regions, known as late assembly (L) domains, which promote the final separation of assembled virions from the cell surface and from each other. The L domain of HIV-1 is in the C-terminal p6gag domain and contains an essential P(T/S)APP motif, whereas the L domains of oncoretroviruses have a more N-terminal location within Gag and a conserved PPXY core motif. L domains can be functionally exchanged between HIV-1 and unrelated retroviruses, indicating that they represent docking sites for cellular interaction partners. Consistent with this view, we recently observed that the L domains of HIV-1 and other viruses induce the ubiquitination of minimal HIV-1 Gag constructs, and that point mutations which abolish L domain activity prevent Gag ubiquitination. Our results imply that the recruitment of the cellular ubiquitination machinery plays a crucial role in L domain function. Our results also indicate that the HIV-1 Gag determinants which engage the ubiquitination system are more complex than those in oncoretroviruses. Based on our preliminary results, we propose to map these determinants in HIV-1 Gag in order to clarify the relationship between Gag ubiquitination and virus release, and to define potential interaction sites for host factors involved in these events. We also propose to investigate the requirement for specific determinants in ubiquitin and the role of specific ubiquitin ligases. Furthermore, we plan to examine whether Gag itself needs to become ubiquitinated, and whether there is a cellular target for L domain-induced ubiquitination. Because our preliminary results show an L domain-dependent enrichment of a 42 kDa protein in Gag immunoprecipitates and in virus-like particles, we also intend to investigate the role of the actin cytoskeleton in HIV-1 L domain function. An understanding of the mechanism by which the HIV-1 L domain promotes virus release may ultimately provide a basis for attempts to block this stage of the HIV-1 replication cycle.