Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes.

The HIV-1 Gag matrix (MA) domain mediates the localization of Gag to the plasma membrane (PM), the site for infectious virion assembly. The MA highly basic region (MA-HBR) interacts with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific acidic lipid. The MA-HBR also binds RNAs. To test whether acidic lipids alone determine PM-specific localization of Gag or whether MA-RNA binding also plays a role, we compared a panel of MA-HBR mutants that contain two types of substitutions at MA residues 25 and 26 or residues 29 and 31: Lys→Arg (KR) (25/26KR and 29/31KR) and Lys→Thr (KT) (25/26KT and 29/31KT). Consistent with the importance of the HBR charge in RNA binding, both KT mutants failed to bind RNA via MA efficiently, unlike the corresponding KR mutants. Both 25/26KT Gag-yellow fluorescent protein (YFP) and 29/31KT Gag-YFP bound nonspecifically to the PM and intracellular membranes, presumably via the myristoyl moiety and remaining MA basic residues. In contrast, 25/26KR Gag-YFP bound specifically to the PM, suggesting a role for the total positive charge and/or MA-bound RNA in navigating Gag to the PM. Unlike 29/31KT Gag-YFP, 29/31KR Gag-YFP was predominantly cytosolic and showed little intracellular membrane binding despite having a higher HBR charge. Therefore, it is likely that MA-RNA binding blocks promiscuous Gag membrane binding in cells. Notably, the introduction of a heterologous multimerization domain restored PI(4,5)P2-dependent PM-specific localization for 29/31KR Gag-YFP, suggesting that the blocking of PM binding is more readily reversed than that of intracellular membrane binding. Altogether, these cell-based data support a model in which MA-RNA binding ensures PM-specific localization of Gag via suppression of nonspecific membrane binding.