Conditional Trimerization and Lytic Activity of HIV-1 gp41 Variants Containing the Membrane-Associated Segments

Abstract

Fusion of host and viral membranes is a critical step during infection by membrane-bound viruses. The HIV-1 glycoproteins gp120 (surface subunit) and gp41 (fusion subunit) represent the prototypic system for studying this process; in the prevailing model, the gp41 ectodomain forms a trimeric six-helix bundle that constitutes a critical intermediate and provides the energetic driving for overcoming barriers associated with membrane fusion. However, most structural studies of gp41 variants have been performed either on ectodomain constructs lacking one or more of membrane-associated segments (the fusion peptide, FP, the membrane-proximal external region, MPER, and transmembrane domain, TM), or on variants consisting of these isolated segments alone without the ectodomain. Several recent reports have suggested that the HIV-1 ectodomain, as well as larger construct containing the membrane-bound segments, dissociate from a trimer to a monomer in detergent micelles. Here we compare the properties of a series of gp41 variants to delineate the roles of the ectodomain, FP, and MPER and TM, all in membrane-mimicking environments. We find that these proteins are prone to formation of a monomer in detergent micelles. In one case, we observed exclusive monomer formation at pH 4 but conditional trimerization at pH 7 even at low micromolar (~5 μM) protein concentrations. Liposome release assays demonstrate that these gp41-related proteins have the capacity to induce content leakage, but that this activity is also strongly modulated by pH with much higher activity at pH 4. Circular dichroism, NMR, and binding assays with antibodies specific to the MPER provide insight into the structural and functional roles of the FP, MPER, and TM and their effect on structure within the larger context of the fusion subunit.

Full-length trimeric influenza virus hemagglutinin II membrane fusion protein and shorter constructs lacking the fusion peptide or transmembrane domain: Hyperthermostability of the full-length protein and the soluble ectodomain and fusion peptide make significant contributions to fusion of membrane vesicles

“…Monomer species exist for both gp41 and HA2 proteins and may be functionally important because folding of the individual protein monomers to the final hairpin state is topologically more straightforward than folding of trimers. 36,41–44 The monomer is dominant for gp41 at pH 3–4 whereas the trimer is dominant for HA2 at pH 7.4 (Fig. 5).…”

The C34 Peptide Fusion Inhibitor Binds to the Six-Helix Bundle Core Domain of HIV-1 gp41 by Displacement of the C-Terminal Helical Repeat Region

“…All experiments were conducted under nearly physiological conditions in 10 mM Tris-HCl buffer (pH 7.6) and 150 mM NaCl, ideal for stable trimerization of gp41, 56 as well as for direct comparison with our earlier binding studies of bivalent and single-chain antibodies directed to the N-HR region of gp41 model proteins. 44 6-helix and 5-helix serve as good models for assessing the binding of the exogenously added C34 peptide by SEC–MALS.…”

Tilted, Uninterrupted, Monomeric HIV-1 gp41 Transmembrane Helix from Residual Dipolar Couplings

“…7,8 However, a recent cryo-EM study of Env, lacking CT but including MPER and TM, did not show electron density for TM, 5 and analytical centrifugation studies of a construct containing MPER and TM (residues 666–715) at pH 7 in dodecylphosphocholine (DPC) micelles showed the peptide to be monomeric. 9 On the other hand, weak trimerization propensity was demonstrated for the TM of the analogous paramyxovirus fusion protein. 10 Recently a structural model for isotopically labeled gp41-TM in small bicelles has been reported, based on NMR data.…”