Trimer Enhancement Mutation Effects on HIV-1 Matrix Protein Binding Activities

Abstract

The HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein cytoplasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA mutations that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus particles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation. T he matrix (MA) domain of the human immunodeficiency type 1 (HIV-1) precursor Gag (Pr55Gag) protein serves several assembly-related functions. One role is to direct Pr55Gag proteins to assembly sites at cell plasma membranes (PMs) that are enriched for phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2) and cholesterol (1-8). This function is accomplished in part by virtue of an amino-terminal myristate moiety and, in part, through direct binding to PI(4,5)P2 (1). Evidence indicates that the PI(4,5)P2-binding site on MA overlaps a binding site for RNA, which suggests a chaperone model in which MA-RNA binding protects the MA domain of Pr55Gag from associating with inappropriate intracellular membranes prior to delivery to the PI(4,5)P2-rich PM assembly sites (9-16). In some experimental systems, it has been shown that Pr55Gag proteins with MA deletions (⌬MA) or swaps of MA with other membrane binding domains are capable of directing the assembly of conditionally infectious viruses, but in these cases, assembly and replication efficiencies have tended to be compromised (17)(18)(19)(20)(21). IMPORTANCE The HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of EnvA second role of HIV-1 MA is to facilitate the incorporation of envelope (Env) proteins into virions (22-25). How MA accomplishes this is complicated. One confounding issue is that some cell surface proteins can be assembled into HIV-1 particles in what appears to be a passive fashion (26). Indeed, this is how glycoproteins from other viruses can pseudotype with the cores of HIV-1 (17, 27-31). Presumably, PM proteins that localize to HIV-1 assembly sites can be incorporated into viruses as...

Distinct functions for the membrane-proximal ectodomain region (MPER) of HIV-1 gp41 in cell-free and cell–cell viral transmission and cell–cell fusion

“…The W666A and I675A mutations diminished the infectivity of cell-free virions, whereas previous studies have shown that membrane fusion mediated by these Env mutants expressed at the cell surface in the absence of other viral proteins is not affected (52). It may be that the MPER mutations adversely affect Env function in the context of assembled cell-free virions, where interactions between MA and the CT of gp41 (61)(62)(63) can modulate the structure and function of the ectodomain (64 -66). To investigate the influence of the CT on the function of the MPER mutants, stop codons were introduced after positions 712, 727, and 752 in the WT, W666A, and I675A pcDNA3.1-AD8env expression vectors to give the ⌬CT144, ⌬CT129, ⌬CT104 mutants, respectively (Fig.…”

“…Several lines of evidence indicate that the gp41 CT interacts with the MA protein shell beneath the inner leaflet of the viral envelope. In this context, the MA shell may include a hexameric array of MA trimers, the central aperture of the hexamer potentially providing a docking site for the CT (61)(62)(63)84). Mutations in MA such as L49D can destabilize the interaction between gp120 and gp41 on the virion surface; however, Env can be uncoupled from the L49D MA defect by ⌬CT144, and this effect can be reproduced by the Y712S substitution in the…”

Meeting Review: 2018 International Workshop on Structure and Function of the Lentiviral gp41 Cytoplasmic Tail

“…Eric Barklis (Oregon Health and Science University) discussed a long-standing question in the field—does Env directly bind MA, and if so, why are so few Env trimers packaged onto HIV-1 particles? To address these questions, Barklis found that the 62QR HIV-1 MA mutant, reported by the Freed lab to rescue Env incorporation defects [ 18 ], forms trimers more efficiently than WT MA in a UV-crosslinking assay, and is bound more efficiently than WT MA to GST-coupled gp41 CT [ 32 ]. The presence of RNA reduced binding between MA and the gp41 CT, perhaps by diminishing MA trimerization, and inositol hexakisphosphate (IP 6 ) increased MA binding to the gp41 CT, perhaps by increasing the MA trimerization.…”

Analysis of quinolinequinone reactivity, cytotoxicity, and anti-HIV-1 properties

“…Incubation products were fractionated on 12% SDS-PAGE gels under reducing conditions (27–8) in parallel with molecular weight size standards, and immunoblotted for MA detection as described previously (27–8), using a primary mouse monoclonal antibody to MA (Capricorn #01848170) at a 1:2000 dilution, an alkaline phosphatase-conjugated anti-mouse secondary antibody (Promega #S3728) at a 1:15,000 dilution, and nitro-blue tetrazolium (NBT; Promega) plus 5-bromo-4-chloro-3'-indolyphosphate (BCIP; Promega) for visualization (27–8). Blots were scanned on and Epson Perfection 1240U scanner, band intensities were obtained with Image J software, and percentage crosslinking levels were determined from dimer divided by dimer plus monomer MA band intensities.…”