Disruption of helix-capping residues 671 and 674 reveals a role in HIV-1 entry for a specialized hinge segment of the membrane proximal external region of gp41

Abstract

HIV-1 (human immunodeficiency virus type 1) uses its trimeric gp160 envelope (Env) protein consisting of non-covalently associated gp120 and gp41 subunits to mediate entry into human T lymphocytes. A facile virus fusion mechanism compensates for the sparse Env copy number observed on viral particles and includes a 22-amino-acid, lentivirus-specific adaptation at the gp41 base (amino acid residues 662-683), termed the membrane proximal external region (MPER). We show by NMR and EPR that the MPER consists of a structurally conserved pair of viral lipid-immersed helices separated by a hinge with tandem joints that can be locked by capping residues between helices. This design fosters efficient HIV-1 fusion via interconverting structures while, at the same time, affording immune escape. Disruption of both joints by double alanine mutations at Env positions 671 and 674 (AA) results in attenuation of Env-mediated cell-cell fusion and hemifusion, as well as viral infectivity mediated by both CD4-dependent and CD4-independent viruses. The potential mechanism of disruption was revealed by structural analysis of MPER conformational changes induced by AA mutation. A deeper acyl chain-buried MPER middle section and the elimination of cross-hinge rigid-body motion almost certainly impede requisite structural rearrangements during the fusion process, explaining the absence of MPER AA variants among all known naturally occurring HIV-1 viral sequences. Furthermore, those broadly neutralization antibodies directed against the HIV-1 MPER exploit the tandem joint architecture involving helix capping, thereby disrupting hinge function.

Keywords: NMR solution structure; broadly neutralizing antibody; helix capping; helix–hinge–helix motif; viral membrane fusion.

Fig. 1

Comparison of MPER segments from 9 groups of lentiviruses. The 23 amino acid long lentiviral segments N-terminal to their respective annotated transmembrane helices were extracted from SwissProt Database. (a) Sequence alignment with colored residues being identical to the reference sequence (HIV-1 HxB2). (b) Logos showing conservation within each of the 9 lentivirus groups. The extremely conserved 3,650 SIV sequences from rhesus macaque (SIV-MAC) is a result of infection in research primate centers.

Fig. 2

Sequence conservation and variation of HIV-1 MPER. (a) Amino acid sequences of MPER peptides used for structural studies, with conserved residues colored in red. (b) HxB2 MPER structure in a DPC detergent micelle with conserved and variable residues colored according to the scale shown. (c) Population of amino acid combinations at the 671 and 674 residue positions at the central hinge region, with BlockLogo sequence conservation diagram of the 671–674 segment shown as an insert at the top with conventional Shannon entropy representation of individual amino acid position variability at the bottom.

Fig. 3

HIV-1 pseudovirus infectivity affected by AA mutations. (a) Infectivity of pseudoviruses harboring the Env mutations at positions 671 and 674. Comparable number of viral particles were used as determined by p24 Elisa. (b) Titration (log₁₀) of Con089 and HxB2 Env WT or mutant pseudoviruses on TZM-bl cells. Mean and standard deviation (sd) of each dilution is shown. (c) Dose dependent infectivity of CD4-independent ADA/Hx(197) pseudoviruses on CD4⁺ CCR5⁺ TZM-bl cells and CD4⁻ Cf2Th/ Syn CCR5 cells. The insert shows anti-gp120 mAb Western-blot of ADA/Hx(197) Env from pseudovirus.

Fig. 4

Env-mediated fusion impaired by AA mutations. (a) Qualitative microscopy analysis of cell-cell fusion. Content mixing of Env-expressing 293T effector cells with 3T3.CD4.CCR5 target cells. The top row shows the overlay of fluorescence images after co-incubation of 293T cells (Green, Calcein) with 3T3.CD4.CCR5 cells (Red, CMTMR) at 37°C for 2 h. The bottom row shows representative bright-field images collected 24 h after co-incubation at 37°C. (b) Fusion kinetics of JR-FL (top) or ADA (bottom) Env-expressing 293T cells with TZM-bl cells containing a Tat-driven luciferase reporter. (c) Hemifusion identified by lipid mixing between 293T cells (green) and DiI-labeled 3T3.CD4.CCR5 cells (red). The top row shows the overlay of fluorescence images collected 2 h after co-incubation at 37°C. The bottom row shows the corresponding bright field images. (d) Quantitative lipid mixing efficiency of JRFL-AA relative to WT. Flow-cytometric analysis of lipid-dye transfer between DiO labeled 293T cells and DiD labeled TZM-bl cells were conducted. The percentage lipid mixing activities were determined following the subtraction of background dye redistribution between empty vector-transfected effector and target cells, normalized to that of WT (100%) in three independent experiments.

Fig. 5

Solution structures of MPER peptides. (a) NMR structure ensembles of Con089, Du151.2, ZM197M.PB7, and HxB2-AA MPER peptides in DPC detergent micelles, superimposed by their N-terminal helices (666–672). (b) The additional CHR residues at the N-terminal end of MPER, as represented here by the Con089 peptide in DPC micelles, all adopt a conserved beta-turn stabilized by L661/L662 and W666. (c) Ribbon diagram of a representative HxB2-AA peptide with the side-chains of alanine substituted residues colored in pink. (d) The left panels show JNH RDC values with good correlation between Con089 and Du151.2, but weaker correlation between HxB2 and HxB2-AA. The right panels show difference in JNH RDC values (normalized according to fitted linear correlation parameters) between Con089 and Du151.2, and between HxB2 and HxB2-AA. The errors are derived from NMR amide peak position estimates.

Fig. 6

Structural comparison with MPER AA mutant. (a) EPR membrane immersion depths of Con089, Du151.2, ZM197M.PB7, and HxB2-AA in liposomes. Depth values between −5 Å to 0 Å and larger than 0 Å correspond to lipid head-group region and acyl-chain region, respectively. The white-colored bars indicate complete exposure to aqueous phase (depth < −5 Å). (b) NMR structure models of representative Con089A, Du151.2, ZM197M.PB7, and HxB2-AA peptides on the membrane surface. The lipid head-group and acyl-chain regions are shown in light blue and yellow, respectively. Residues 671 and 674 are colored in pink. The N-terminal extended regions (657–661) are omitted for simplicity. (c) EPR DEER spectra of singly spin-labeled HxB2-AA MPER peptides at residue positions 670 (top), 678 (middle) and 681 (bottom) showing spin-spin correlation consistent with peptide dimerization. (d) NMR ¹⁵N-HSQC spectra of HxB2-AA MPER in DPC micelles, showing broadening and disappearance of some backbone amide peaks at higher peptide concentrations consistent with aggregation.

Fig. 7

MPER conformation change during HIV membrane fusion. (a) Illustrations of the HIV fusion process following CD4 binding. Chemokine receptors are omitted at the pre-fusion stage and only one gp41 monomer is drawn in the intermediate stages for simplicity. MPER is highlighted in red. (b) Hinge conformation revealed by backbone dihedral angles for free and BNAb-bound HxB2 wild-type MPER derived from observed NMR chemical shifts, except for 10E8 where the angles are extracted from a crystal structure (PDB: 4G6F). The backbone dihedral angles extracted from unbound Con089 and HxB2-AA peptide NMR structures are shown for comparison. (c) Side-chain oxygen to backbone amide hydrogen bonds observed in crystal structures of MPER bound to BNAbs: (top) between N671 to D674 in 10E8, (middle) between N671 to F673 in 4E10 (PDB: 2FX7), and (bottom) between D674 to T676 in Z13e1 (PDB: 3FN0). (d) Orientation change of W672/F673 with respect to the membrane with different N-capping of the C-terminal helix in 10E8-bound and unbound MPER.