Cholesterol-Dependent Membrane Fusion Induced by the gp41 Membrane-Proximal External Region–Transmembrane Domain Connection Suggests a Mechanism for Broad HIV-1 Neutralization

Abstract

The HIV-1 glycoprotein 41 promotes fusion of the viral membrane with that of the target cell. Structural, biochemical, and biophysical studies suggest that its membrane-proximal external region (MPER) may interact with the HIV-1 membrane and induce its disruption and/or deformation during the process. However, the high cholesterol content of the envelope (ca. 40 to 50 mol%) imparts high rigidity, thereby acting against lipid bilayer restructuring. Here, based on the outcome of vesicle stability assays, all-atom molecular dynamics simulations, and atomic force microscopy observations, we propose that the conserved sequence connecting the MPER with the N-terminal residues of the transmembrane domain (TMD) is involved in HIV-1 fusion. This junction would function by inducing phospholipid protrusion and acyl-chain splay in the cholesterol-enriched rigid envelope. Supporting the functional relevance of such a mechanism, membrane fusion was inhibited by the broadly neutralizing 4E10 antibody but not by a nonneutralizing variant with the CDR-H3 loop deleted. We conclude that the MPER-TMD junction embodies an envelope-disrupting C-terminal fusion peptide that can be targeted by broadly neutralizing antibodies. IMPORTANCE Fusion of the cholesterol-enriched viral envelope with the cell membrane marks the beginning of the infectious HIV-1 replicative cycle. Consequently, the Env glycoprotein-mediated fusion function constitutes an important clinical target for inhibitors and preventive vaccines. Antibodies 4E10 and 10E8 bind to one Env vulnerability site located at the gp41 membrane-proximal external region (MPER)-transmembrane domain (TMD) junction and block infection.These antibodies display broad viral neutralization, which underscores the conservation and functionality of the MPER-TMD region. In this work, we combined biochemical assays with molecular dynamics simulations and microscopy observations to characterize the unprecedented fusogenic activity of the MPER-TMD junction. The fact that such activity is dependent on cholesterol and inhibited by the broadly neutralizing 4E10 antibody emphasizes its physiological relevance. Discovery of this functional element adds to our understanding of the mechanisms underlying HIV-1 infection and its blocking by antibodies.

The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region

“…However, the Env(671-704) sequence covering the entire gp41 TMD and a fraction of the adjacent MPER shows a high degree of conservation (18,26,36), consistent with the additional functional roles ascribed to this region during the infectious cycle, i.e. membrane fusion promotion (17,18,36) and immune response modulation (30 -32). Despite these important functional roles, high resolution structural data on the gp41 TMD and its junction to MPER were lacking ( Table 4).…”

“…Although the molecular mechanism remains unsolved, MPER-N-TMD promotes membrane fusion as evidenced by mutagenesis studies and peptide-based assays (18,36,64,70). Consistent with a pivotal role in the fusion process, bNAbs raised .…”

“…Following a strategy similar to that described in our previous study (34), those experiments included a Fab4E10 mutant with the CDR-H3 loop tip ablated as a negative control. The Fab4E10-⌬loop mutant binds peptide epitopes in solution with comparable affinity, but it is not neutralizing (36), and therefore allows discriminating functional versus nonfunctional binding.…”

Characterizing the Murine Leukemia Virus Envelope Glycoprotein Membrane-Spanning Domain for Its Roles in Interface Alignment and Fusogenicity

“…Similarly, the membrane-proximal external region (MPER) and MSD of HIV-1 Env have been shown to be crucial for promoting fusogenicity. Disruption of hinge regions or the tryptophan-rich sequence in the MPER of HIV-1 Env, as well as polar residues within the MSD, all result in nonfunctional glycoproteins that are fusogenically inactive (24)(25)(26)(27)(28)(29). The potential role for the MSDs of MLV and HIV-1 Env in potentiating molecular rearrangements necessary for fusogenic activity would be analogous to the MSDs of voltage-gated ion channels and G-protein-coupled receptors (GPCRs).…”

In Vivo Analysis of Infectivity, Fusogenicity, and Incorporation of a Mutagenic Viral Glycoprotein Library Reveals Determinants for Virus Incorporation

“…Even though glycoproteins are not observed to cluster in large preexisting domains in the absence of viral assembly, viral assembly could trigger the coalescence of smaller microdomains that contain the viral glycoproteins. It has been demonstrated that the cholesterol recognition amino acid consensus (CRAC) domain in the membrane-proximal external region (MPER) of HIV-1 Env has a propensity to interact with cholesterol-rich domains in the plasma membrane (25)(26)(27)(28)(29) and is necessary for remodeling of the HIV-1 lipid membrane to facilitate fusion (30)(31)(32). Additionally, the tryptophan-rich region immediately upstream of the HIV-1 Env CRAC domain has been implicated in HIV-1 Env acquisition into viral particles (30).…”