Virus particle assembly of HIV-1, the causative agent of AIDS, takes place at the plasma membrane (PM) in most cell types including natural host T cells. This process is driven by a viral structural protein Gag. The N-terminal matrix (MA) domain of Gag determines Gag localization to and hence virus assembly at the PM. MA mediates membrane binding of Gag via N-terminal myristoyl moiety and a highly basic region (HBR) that binds acidic lipids. Binding of HBR to a PM-specific acidic phospholipid PI(4,5)P2 is critical for PM localization of Gag and efficient virus release. Notably, we and others showed that MA HBR also interacts with tRNAs, which suppress binding of Gag to non-PI(4,5)P2 acidic lipids, suggesting tRNAs as a host factor that regulates MA-membrane interactions. However, structural determinants for the tRNA-MA HBR interaction and its reversal by the interaction with PI(4,5)P2, combination of which regulates PM-specific Gag localization, remain to be examined. Binding of tRNAs to MA HBR is most likely to occur at translation sites due to limited availability of tRNAs outside of the translation machinery. However, little is known about subcellular sites of Gag translation, where Gag begins its movement to the PM. At the PM, Gag multimerization and subsequent accumulation of acidic lipids are likely to promote recruitment of host transmembrane proteins, but their effects on virus spread to uninfected cells remains to be determined in the context of cell-free and cell-to-cell transmission. Our long-term goal is to elucidate mechanisms that determine sites of HIV-1 assembly and to use the knowledge for developing antiviral strategies. Our central hypothesis in this application is that MA HBR interactions with tRNAs, which begin during translation, and with acidic lipids determine subcellular Gag localization and the properties of progeny virions. To test this hypothesis, we plan to: 1) identify structural determinants for interactions of MA HBR with tRNAs and acidic lipids; 2) identify tRNAs that suppress PI(4,5)P2-independent membrane binding but allow PI(4,5)P2-mediated reversal; 3) understand the effects of Gag translation sites on the fate of Gag; and 4) examine the effects of host transmembrane proteins incorporated into virus particles on formation of virological synapse and virus-cell contact. The knowledge gained from experiments outlined in this proposal will likely help us develop antiviral strategies that target mechanisms regulating Gag localization to the PM, thereby inhibiting extracellular virus release and spread.