Assembly of HIV-1 particles is initiated by the trafficking of viral Gag polyproteins from the cytoplasm to the plasma membrane (PM), 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 PM 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 (CA), 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, supporting proposals that Gag binding may nucleate raft formation. 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.

The N-terminally myristoylated matrix (MA) domain of the HIV-1 Gag polyprotein promotes virus assembly by targeting Gag to the inner leaflet of the plasma membrane. Recent studies indicate that, prior to membrane binding, MA associates with cytoplasmic tRNAs (including tRNA), and in vitro studies of tRNA-dependent MA interactions with model membranes have led to proposals that competitive tRNA interactions contribute to membrane discrimination. We have characterized interactions between native, mutant, and unmyristylated (myr-) MA proteins and recombinant tRNA by NMR spectroscopy and isothermal titration calorimetry. NMR experiments confirm that tRNA interacts with a patch of basic residues that are also important for binding to the plasma membrane marker, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P]. Unexpectedly, the affinity of MA for tRNA (K = 0.63 ± 0.03 μM) is approximately 1 order of magnitude greater than its affinity for PI(4,5)P-enriched liposomes (K = 10.2 ± 2.1 μM), and NMR studies indicate that tRNA binding blocks MA association with liposomes, including those enriched with PI(4,5)P, phosphatidylserine, and cholesterol. However, the affinity of MA for tRNA is diminished by mutations or sample conditions that promote myristate exposure. Since Gag-Gag interactions are known to promote myristate exposure, our findings support virus assembly models in which membrane targeting and genome binding are mechanistically coupled.

The matrix (MA) domain of the HIV-1 Gag is responsible for Gag targeting to the plasma membrane where virions assemble. MA also plays a role in the incorporation of the viral envelope (Env) glycoproteins, and can influence particle infectivity post-maturation and post-entry. A highly basic region of MA targets Gag to the plasma membrane via specific binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. This binding also triggers exposure of an amino-terminal myristate moiety, which anchors Gag to the membrane. A MA mutant deficient for PI(4,5)P2 binding, 29KE/31KE, has been shown to mislocalize within the cell, leading to particle assembly in a multivesicular body compartment and defective release of cell-free particles in HeLa and 293T cells. Despite the defect in virus production in these cells, release of the 29KE/31KE mutant is not significantly reduced in primary T cells, macrophages, and Jurkat T cells. 29KE/31KE virions also display an infectivity defect associated with impaired Env incorporation, irrespective of the producer cell line. Here we examine the properties of 29KE/31KE by analyzing compensatory mutations obtained by a viral adaptation strategy. The MA mutant 16EK restores virus release through enhanced membrane binding. 16EK also influences the infectivity defect, in combination with an additional MA mutant, 62QR. Additionally, the 29KE/31KE MA mutant displays a defect in proteolytic cleavage of the murine leukemia virus Env cytoplasmic tail in pseudotyped virions. Our findings elucidate the mechanism whereby a MA mutant defective in PI(4,5)P2 binding can be rescued and highlight the ability of MA to influence Env glycoprotein function.