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HIV-1 gp41 transmembrane domain interacts with the fusion peptide: implication in lipid mixing and inhibition of virus-cell fusion.

ABSTRACT: Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion peptide (FP) at the late fusion stages, but there are no direct experimental data to support this hypothesis. Here, we investigated the TMD focusing on two regions: the N-terminal containing the GxxxG motif and the C-terminal containing the GLRI motif, which is conserved among the TMDs of HIV and the T-cell receptor. Studies utilizing the ToxR expression system combined with synthetic peptides and their fluorescent analogues derived from TMD revealed that the GxxxG motif is important for TMD self-association, whereas the C-terminal region is for its heteroassociation with FP. Functionally, all three TMD peptides induced lipid mixing that was enhanced significantly upon mixing with FP. Furthermore, the TMD peptides inhibited virus-cell fusion apparently through their interaction with their endogenous counterparts. Notably, the R2E mutant (in the GLRI) was significantly less potent than the two others. Overall, our findings provide experimental evidence that HIV-1 TMD contributes to membrane assembly and function of the HIV-1 envelope. Owing to similarities between functional domains within viruses, these findings suggest that the TMDs and FPs may contribute similarly in other viruses as well.

SUBMITTER: Reuven EM

PROVIDER: S-EPMC3335273 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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HIV-1 gp41 transmembrane domain interacts with the fusion peptide: implication in lipid mixing and inhibition of virus-cell fusion.

Reuven Eliran Moshe EM Dadon Yakir Y Viard Mathias M Manukovsky Nurit N Blumenthal Robert R Shai Yechiel Y

Biochemistry 20120323 13

Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion peptide (FP) at the late fusion stages, but there are no direct experimental data to support this hypothesis. Here, we investigated the TMD focusing on two regions: the N-terminal ...[more]

PMID: 22413880

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Project description:Human immunodeficiency virus (HIV) is membrane-enveloped, and an initial infection step is joining/fusion of viral and cell membranes. This step is catalyzed by gp41, which is a single-pass integral viral membrane protein. The protein contains an ?170-residue ectodomain located outside the virus that is important for fusion and includes the fusion peptide (FP), N-helix, loop, C-helix, and viral membrane-proximal external region (MPER). The virion initially has noncovalent complexes between three gp41 ectodomains and three gp120 proteins. A gp120 contains ?500 residues and functions to identify target T-cells and macrophages via binding to specific protein receptors of the target cell membrane. gp120 moves away from the gp41 ectodomain, and the ectodomain is thought to bind to the target cell membrane and mediate membrane fusion. The secondary and tertiary structures of the ectodomain are different in the initial complex with gp120 and the final state without gp120. There is not yet imaging of gp41 during fusion, so the temporal relationship between the gp41 and membrane structures is not known. This study describes biophysical and functional characterization of large gp41 constructs that include the ectodomain and transmembrane domain (TM). Significant fusion is observed of both neutral and anionic vesicles at neutral pH, which reflects the expected conditions of HIV/cell fusion. Fusion is enhanced by the FP, which in HIV/cell fusion likely contacts the host membrane, and the MPER and TM, which respectively interfacially contact and traverse the HIV membrane. Initial contact with vesicles is made by protein trimers that are in a native oligomeric state that reflects the initial complex with gp120 and also is commonly observed for the ectodomain without gp120. Circular dichroism data support helical structure for the N-helix, C-helix, and MPER and nonhelical structure for the FP and loop. Distributions of monomer, trimer, and hexamer states are observed by size-exclusion chromatography (SEC), with dependences on solubilizing detergent and construct. These SEC and other data are integrated into a refined working model of HIV/cell fusion that includes dissociation of the ectodomain into gp41 monomers followed by folding into hairpins that appose the two membranes, and subsequent fusion catalysis by trimers and hexamers of hairpins. The monomer and oligomer gp41 states may therefore satisfy dual requirements for HIV entry of membrane apposition and fusion.

Project description: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.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.

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HIV-1 gp41 transmembrane domain interacts with the fusion peptide: implication in lipid mixing and inhibition of virus-cell fusion.

Publications

HIV-1 gp41 transmembrane domain interacts with the fusion peptide: implication in lipid mixing and inhibition of virus-cell fusion.

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