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. 1999 Oct;73(10):8578-86.
doi: 10.1128/JVI.73.10.8578-8586.1999.

Inhibition of human immunodeficiency virus type 1 infectivity by the gp41 core: role of a conserved hydrophobic cavity in membrane fusion

H Ji  1 , W Shu, F T Burling, S Jiang, M Lu
Affiliations

Inhibition of human immunodeficiency virus type 1 infectivity by the gp41 core: role of a conserved hydrophobic cavity in membrane fusion

H Ji et al. J Virol. 1999 Oct.

Abstract

The gp41 envelope protein of human immunodeficiency virus type 1 (HIV-1) contains an alpha-helical core structure responsible for mediating membrane fusion during viral entry. Recent studies suggest that a conserved hydrophobic cavity in the coiled coil of this core plays a distinctive structural role in maintaining the fusogenic conformation of the gp41 molecule. Here we investigated the importance of this cavity in determining the structure and biological activity of the gp41 core by using the N34(L6)C28 model. The high-resolution crystal structures of N34(L6)C28 of two HIV-1 gp41 fusion-defective mutants reveal that each mutant sequence is accommodated in the six-helix bundle structure by forming the cavity with different sets of atoms. Remarkably, the mutant N34(L6)C28 cores are highly effective inhibitors of HIV-1 infection, with 5- to 16-fold greater activity than the wild-type molecule. The enhanced inhibitory activity by fusion-defective mutations correlates with local structural perturbations close to the cavity that destabilize the six-helix bundle. Taken together, these results indicate that the conserved hydrophobic coiled-coil cavity in the gp41 core is critical for HIV-1 entry and its inhibition and provides a potential antiviral drug target.

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Figures

FIG. 1
FIG. 1
Schematic diagram of HIV-1 gp41. The important functional features of the gp41 ectodomain and the amino acid sequences of the N34 and C28 segments are shown. The N34(L6)C28 model consists of N34 and C28 plus a six-residue linker. Two point mutations, L568A and W571R, that abolish membrane fusion are indicated above the sequence. The disulfide bond and four potential N-glycosylation sites are depicted. The residues are numbered according to their position in gp160.
FIG. 2
FIG. 2
Folding of the N34(L6)C28 mutants as helical trimers. (A) CD spectra of L568A (squares), W571R (triangles), and L568A/W571R (circles) at 0°C in PBS (pH 7.0) at a peptide concentration of 10 μM. (B) Thermal melts monitored by CD at 222 nm for L568A (squares), W571R (triangles), and L568A/W571R (circles) in PBS (pH 7.0) at a peptide concentration of 10 μM. (C) Sedimentation equilibrium studies of the mutant N34(L6)C28 cores indicate that all species are trimeric. Representative analytical ultracentrifugation data (20 krpm) for L568A/W571R collected at 20°C in PBS (pH 7.0) at a peptide concentration of ∼30 μM are shown. The natural logarithm of the absorbance at 280 nm is plotted against the square of the radial position. Deviations from the calculated values are plotted as residuals in the upper panel.
FIG. 3
FIG. 3
Electron density maps at the substitution sites. (A) Initial 2Fo − Fc map of L568A after density modification and phase improvement with DM (14). The initial molecular replacement solution model is superimposed. (B) Final 2Fo − Fc map of L568A with the refined model superimposed. (C) Initial 2Fo − Fc map of W571R as described for panel A. (D) Final 2Fo − Fc map of W571R as described for panel B. The side chains of the mutated residues are indicated by white arrows. Water molecules are indicated by small red balls. The maps of L568A and W571R are contoured at 1.8 and 1.2 ς, respectively. Figures were generated with the program O (33).
FIG. 4
FIG. 4
Overall views of the mutant L568A and W571R cores. (A) Side view. The amino termini of N34 and the carboxyl termini of C28 are at the top. Helices in L568A (yellow) and W571R (pink) were used for the superposition. The bottom of the central N34 coiled-coil surface contains three symmetry-related hydrophobic cavities (one is outlined by the box). (B) View from the top, looking down the threefold axis of the trimer. The same color coding as in panel A is used. Figures were generated with the program SETOR (21).
FIG. 5
FIG. 5
The conserved hydrophobic cavity in the L568A and W571R structures. (A) Cross-section of helix packing near the conserved cavity in L568A. The structures of the wild-type molecule (red) and L568A (green) are overlaid. The side chains of the mutated residues are in yellow. (B) Cross-section of helix packing in W571R. The same superposition as in panel A was used. (C) Interactions of the C28 helix with a deep cavity on the surface of the N34 coiled coil in L568A. The C28 helices of the wild-type molecule (red) and L568A (green), represented as ribbons, are shown against a surface representation of the N34 coiled coil in L568A. (D) Interactions of the C28 helix with a deep cavity on the surface of the N34 coiled coil in W571R. The same superposition as in panel C was used. Figures were generated with the programs SETOR (21) and GRASP (47).
FIG. 6
FIG. 6
Inhibition of HIV-1 infection by N34(L6)C28 variants. (A) Inhibition of HIV-1IIIB-infected H9 cell-induced cell-cell fusion by N34(L6)C28 (diamonds), L568A (triangles), W571R (squares), and L568A/W571R (circles). Error bars indicate standard deviations from quadruplicate experiments. (B) Inhibition of HIV-1IIIB-mediated CPE by N34(L6)C28 (diamonds), L568A (triangles), W571R (squares), and L568A/W571R (circles). Error bars indicate standard deviations from triplicate experiments.
FIG. 7
FIG. 7
An Ile 573-to-Ser mutation disrupts the six-helix bundle formation of L568A/W571R and abolishes its potent antiviral activity. (A) CD spectrum of L568A/W571R/I573S at 0°C in PBS (pH 7.0) at a peptide concentration of 10 μM. The inset shows a thermal melt monitored by CD at 222 nm for L568A/W571R/I573S in PBS (pH 7.0) at a peptide concentration of 10 μM. (B) Inhibition of HIV-1IIIB-infected H9 cell-induced cell-cell fusion (circles) and HIV-1IIIB-mediated CPE (triangles) by L568A/W571R/I573S. Error bars indicate standard deviations from quadruplicate and triplicate experiments for cell-cell fusion and infectivity assays, respectively.
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