Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

Abstract

By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals-electrostatic, hydrophobic, and lipid-specific-to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ⌬G, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P 2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P 2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P 2 , the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P 2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCELike other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA. T he polyprotein Gag is an essential component for the reproduction of retroviruses, such as HIV-1, in infected cells. In the production of new viruses, many copies of Gag assemble laterally, either in the cytoplasm or on the plasma membrane (PM) of the host, thus acquiring their lipid envelope as they bud from the cell. Distinct retroviral genera encode Gag proteins which all show a similar domain architecture, with an N-terminal matrix (MA) domain that targets the PM, followed by the capsid (CA) and nucleocapsid ...

Self assembly of HIV-1 Gag protein on lipid membranes generates PI(4,5)P2/Cholesterol nanoclusters

“…However, it is known that cholesterol is crucial for virus infectivity3637 and that Gag can sense cholesterol3839. It was recently described that PIP 2 could form clusters in the presence of cholesterol alone40.…”

HIV-1 matrix-31 membrane binding peptide interacts differently with membranes containing PS vs. PI(4,5)P2

“…Self-assembled monolayers (SAMs) of HC18 tethers were formed on 3” diameter silicon wafers as previously described [32]. Sparsely-tethered bilayer lipid membranes (stBLMs) were formed by exposing the SAM to the vesicle suspension for 60 minutes in a NIST reflectivity flow cell, followed by a rinse with 40 ml deionized water [28]. …”

“…A recent SPR study examined the role of lipids in membrane binding of the myristoylated and non-myristoylated forms of MA [28] and reported that myristoylation increases MA affinity to PC/PS membranes by factors up to ~20 depending on membrane charge. Cholesterol in PC/PS membranes increased MA affinity only mildly, while it increased it much more in PIP 2 -containing PC/PS membranes, in particular for the myristoylated protein.…”

“…Cholesterol in PC/PS membranes increased MA affinity only mildly, while it increased it much more in PIP 2 -containing PC/PS membranes, in particular for the myristoylated protein. This suggests yet another additive effect of MA protein binding, between the myristoyl group and PIP 2 [28]. Therefore, membrane binding is a sensitive assay of the contributions of different lipids to the MA protein/membrane interaction; however, binding is only one part of the picture.…”

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

“… Note Added in Proof After this paper was submitted and reviewed, a preprint appeared on-line describing studies of MA binding to synthetic solid-supported bilayers by surface plasmon resonance spectroscopy [113]. …”