Opposing mechanisms involving RNA and lipids regulate HIV-1 Gag membrane binding through the highly basic region of the matrix domain

Membrane binding of Gag, a crucial step in HIV-1 assembly, is facilitated by bipartite signals within the matrix (MA) domain: N-terminal myristoyl moiety and the highly basic region (HBR). We and others have shown that Gag interacts with a plasma-membrane-specific acidic phospholipid, phosphatidylinositol(4,5)-bisphosphate [PI(4,5)P-2], via the HBR, and that this interaction is important for efficient membrane binding and plasma membrane targeting of Gag. Generally, in protein-PI(4,5)P-2 interactions, basic residues promote the interaction as docking sites for the acidic headgroup of the lipid. In this study, toward better understanding of the Gag-PI(4,5)P-2 interaction, we sought to determine the roles played by all of the basic residues in the HBR. We identified three basic residues promoting PI(4,5)P-2-dependent Gag-membrane binding. Unexpectedly, two other HBR residues, Lys25 and Lys26, suppress membrane binding in the absence of PI(4,5)P-2 and prevent promiscuous intracellular localization of Gag. This inhibition of nonspecific membrane binding is likely through suppression of myristate-dependent hydrophobic interaction because mutating Lys25 and Lys26 enhances binding of Gag with neutral-charged liposomes. These residues were reported to bind RNA. Importantly, we found that RNA also negatively regulates Gag membrane binding. In the absence but not presence of PI(4,5)P-2, RNA bound to MA HBR abolishes Gag-liposome binding. Altogether, these data indicate that the HBR is unique among basic phosphoinositide-binding domains, because it integrates three regulatory components, PI(4,5)P-2, myristate, and RNA, to ensure plasma membrane specificity for particle assembly.