The specific interface for selenocysteine-decoding tRNA in HIV-1 Gag MA domain regulates progeny virion infectivity

Localization of HIV-1 particle assembly at the plasma membrane (PM) is mediated by the HIV-1 Gag matrix (MA) domain. Competing binding of PM acidic phospholipids and cellular tRNAs to MA-highly basic region (MA-HBR) likely regulate the PM-specific binding of Gag; however, the specificity of the MA-tRNA interaction and its role in viral replication remain poorly understood. Here, using a modified crosslinking-immunoprecipitation (CLIP)-sequencing workflow for identifying MA-bound tRNAs, we determined that most MA-bound tRNAs were also abundantly present in cells. However, selenocysteine-tRNA (SeCTCA) is overrepresented among Gag-bound tRNAs despite its paucity. The binding of SeCTCA to Gag is entirely dependent on MA-HBR, whereas non-SeCTCA tRNAs can bind Gag through both MA and nucleocapsid domains. Structure prediction of MA-tRNA complexes revealed the binding interface unique to the MA-SeCTCA complex, which was verified experimentally. CLIP experiments demonstrated that this interface is specifically needed for the MA binding to SeCTCA but not to other tRNAs. Notably, disruption of this interface altered Gag localization pattern, reduced infectivity of virions, and impaired virus replication in CD4+ T cells. Collectively, our results indicate that SeCTCA tRNA binds the HIV-1 Gag MA domain with high specificity and promotes virion formation and efficient replication in T cells.