Structural and Molecular Determinants of Membrane Binding by the HIV-1 Matrix Protein

Abstract

Assembly of HIV-1 particles is initiated by the trafficking of viral Gag polyproteins from the cytoplasm to the plasma membrane, where they co-localize and bud to form immature particles. Membrane targeting is mediated by the N-terminally myristoylated matrix (MA) domain of Gag and is dependent on the plasma membrane marker phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. Recent studies revealed that PI(4,5)P₂ molecules containing truncated acyl chains [tr-PI(4,5)P₂] are capable of binding MA in an “extended lipid” conformation and promoting myristoyl exposure. Here we report that tr-PI(4,5)P₂ molecules also readily bind to non-membrane proteins, including HIV-1 capsid, which prompted us to re-examine MA–PI(4,5)P₂ interactions using native lipids and membrane mimetic liposomes and bicelles. Liposome binding trends observed using a recently developed NMR approach paralleled results of flotation assays, although the affinities measured under the equilibrium conditions of NMR experiments were significantly higher. Native PI(4,5)P₂ enhanced MA binding to liposomes designed to mimic non-raft-like regions of the membrane, suggesting the possibility that binding of the protein to disordered domains may precede Gag association with, or nucleation of, rafts. Studies with bicelles revealed a subset of surface and myr-associated MA residues that are sensitive to native PI(4,5)P₂, but cleft residues that interact with the 2'-acyl chains of tr-PI(4,5)P₂ molecules in aqueous solution were insensitive to native PI(4,5)P₂ in bicelles. Our findings call to question extended-lipid MA:membrane binding models, and instead support a model put forward from coarse-grained simulations indicating that binding is mediated predominantly by dynamic, electrostatic interactions between conserved basic residues of MA and multiple PI(4,5)P₂ and phosphatidylserine molecules.