Prolonged exposure of the HIV-1 gp41 membrane proximal region with L669S substitution

Abstract

The conserved membrane-proximal external region (MPER) of HIV-1 envelope is a target for the rare broadly neutralizing 2F5, Z13, and 4E10 monoclonal antibodies (mAbs). One strategy to elicit such antibodies is to design an immunogen with increased exposure of the 2F5 and 4E10 mAb epitopes. In this study we characterize a single leucine to serine substitution at position 669 (L669S) in the gp41 Env MPER that confers >250-fold more neutralization sensitivity to 2F5 and 4E10 mAbs than does the wild-type gp41 sequence. On synthetic liposomes, increased solvent exposure of MPER tryptophan residues and stable docking of 2F5 and 4E10 mAbs to mutant MPER peptide liposomes indicate more favorable membrane orientation of MPER neutralizing epitopes with L669S substitution. The time during which virus is sensitive to 2F5 mAb-mediated neutralization is approximately 3-fold longer when the mutation is present. These data suggest that a major contribution to the L669S mutant virus phenotype of enhanced susceptibility to MPER mAbs is prolonged exposure of the MPER neutralizing epitope during viral entry.

Fig. 1.

Neutralization of pseudotyped viruses containing the L669S mutation. IC₅₀ values of 2F5 mAb and 4E10 mAb neutralization of a primary isolate, QZ4734 containing the L669S mutation (QZ4734/L669S) by site-directed mutagenesis are shown compared to TND_669S and TND_669L.

Fig. 2.

Binding of 2F5 and 4E10 mAbs to gp41 MPER peptide epitopes. Specific binding responses of 2F5 mAb (A and B: 41.6, 83.3, and 167 nM) and 4E10 mAb (C and D: 33.3, 67, 133, 200, 267, and 333 nM) to gp41 MPER_656–683/L669S (A) and gp41MPER_656–683 (B) peptides are shown. The K_d values shown were estimated by global curve fitting of the specific binding responses using a bivalent analyte model. Fitted curves are in black.

Fig. 3.

Effect of L669S mutation on 2F5 epitope exposure for membrane-anchored peptides. Tryptophan fluorescence emission spectra of MPER_652–671 peptide liposomes (A) and MPER_652–671/L669S peptide liposomes (B) at different added acrylamide concentrations are shown. Stern–Volmer plots of quenching experiments shown in A and B are shown in C and D, respectively. F₀/F is the ratio between the intensities of tryptophan fluorescence in the absence of and at different concentrations of acrylamide. a.u, arbitrary unit.

Fig. 4.

Binding of 2F5 and 4E10 mAbs and Fabs to peptide–liposome conjugates. Comparison of 2F5 and 4E10 mAb (A and B) and the respective Fab (C and D) binding to MPER_656–683 peptide (red), MPER_{656–683/L669S} peptide (blue), and peptide-free synthetic liposomes (black) is shown. RU, resonance unit.

Fig. 5.

Time course of 2F5 neutralization of TND L669 and TND 669S pseudoviruses. 2F5 mAb (A and B) or T20 peptide (C and D) at inhibitory concentrations (100 μg/mL for TND 669L and 5 μg/mL for TND 669S for 2F5 mAb and 20 μg/mL for both viruses for T20) was added at the indicated time to TZM-bl cells preincubated with either the wild-type TND 669L (A and C) or the mutant TND_669S (B and D) pseudotyped virus. The mean t_1/2 values (±SE) of 2F5 and T20 neutralizations are as follows: 2F5 mAb, 14.5 ± 0.9 (WT TND_669L), 45.1 ± 2.1 (mutant TND_669S); T20, 20.2 ± 0.52 (WT TND_669L), 33.3 ± 3.2 (mutant TND_669S). t_1/2 values were derived from a four-parameter sigmoid curve-fitting analysis (goodness of fit >0.98) and are means of three measurements. Three individual plots are overlaid for 2F5 mAb (A and B) and two for T20 peptide (C and D).