Efficient Fusion at Neutral pH by Human Immunodeficiency Virus gp41 Trimers Containing the Fusion Peptide and Transmembrane Domains

Abstract

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.

The Stabilities of the Soluble Ectodomain and Fusion Peptide Hairpins of the Influenza Virus Hemagglutinin Subunit II Protein Are Positively Correlated with Membrane Fusion

“…The SE hairpin maintains membrane apposition during the ~1 minute fusion time, and clustering of hairpin trimers and therefore FP domains in the target membrane perturbs this membrane and lowers the activation barrier to achieve hemifusion and pore formation. 11, 26, 47, 52 For this model, the SE hairpin is a fusogenic state rather than a post-fusion state. This model is supported by the WT-HA2-induced vesicle fusion at pH 5.0, which is observed with <100 HA2 trimers/vesicle, less than the ~400 HA trimers/influenza virion.…”

“…66, 67 A FP+SE+TM gp41 construct in this final state catalyzes fusion at pH 7.4 between vesicles composed of PC:Cholesterol or PC:PG:Cholesterol. 52 This is the pH of HIV/host-cell fusion, and there are significant fractions of PC and anionic lipids in the plasma membranes of cells infected by HIV. 68 Gp160-cells also fuse with receptor-bearing cells, and the V2E mutation at the N-terminus of gp41 results in highly-impaired V2E- vs. WT- gp160-cell/receptor-cell fusion.…”

“…A monomer fraction for HA2 in SEC correlates with monomer fractions of the initial HA1/HA2 complex under some conditions, as well as large monomer fractions for gp41 under some conditions. 47, 48, 50, 52, 56, 57, 70 For both HA2 and gp41, there is a significant structural rearrangement between the initial trimeric HA1/HA2 or gp160 complex, and the final trimer-of-hairpins without the receptor proteins. Transient dissociation of HA2 and gp41 into monomers may be functionally important because monomer rearrangement into a hairpin followed by association into a trimer-of- hairpins may be topologically more straightforward than a concerted rearrangement of trimeric protein.…”

“…Monomer gp41 may also be the binding target of peptides which inhibit gp160-mediated fusion and HIV infection, and whose sequences correspond to segments of the C-terminal region of the gp41 SE. 52, 63…”

Insights into the mechanism of membrane fusion induced by the plant defense element, plant-specific insert

“…It is commonly accepted that the membrane fusion process involves a dehydration process. Solid-state NMR (SSNMR) is powerful in studying the topology of membrane fusion proteins (21)(22)(23)(24)(25)(26)(27). In particular, the SSNMR based H/D exchange experiments would provide insights to study the solvent accessibility of the protein, thus suggesting the level of hydration.…”