Binding of the mannose-specific lectin, griffithsin, to HIV-1 gp120 exposes the CD4-binding site

Abstract

The glycans on HIV-1 gp120 play an important role in shielding neutralization-sensitive epitopes from antibody recognition. They also serve as targets for lectins that bind mannose-rich glycans. In this study, we investigated the interaction of the lectin griffithsin (GRFT) with HIV-1 gp120 and its effects on exposure of the CD4-binding site (CD4bs). We found that GRFT enhanced the binding of HIV-1 to plates coated with anti-CD4bs antibodies b12 and b6 or the CD4 receptor mimetic CD4-IgG2. The average enhancement of b12 or b6 binding was higher for subtype B viruses than for subtype C, while for CD4-IgG2, it was similar for both subtypes, although lower than observed with antibodies. This GRFT-mediated enhancement of HIV-1 binding to b12 was reflected in synergistic neutralization for 2 of the 4 viruses tested. The glycan at position 386, which shields the CD4bs, was involved in both GRFT-mediated enhancement of binding and neutralization synergism between GRFT and b12. Although GRFT enhanced CD4bs exposure, it simultaneously inhibited ligand binding to the coreceptor binding site, suggesting that GRFT-dependent enhancement and neutralization utilize independent mechanisms. This study shows for the first time that GRFT interaction with gp120 exposes the CD4bs through binding the glycan at position 386, which may have implications for how to access this conserved site.

Fig. 1.

HIV-1 capture by MAbs b12 and b6 was enhanced in the presence of GRFT. HIV-1 env-pseudotyped viruses from subtype B (QH0692.42 and PVO.4) and subtype C (COT6.15, COT9.6, Du151.2, and CAP239.G3J) were incubated with three different concentrations of GRFT before being added to plates coated with either b12 (A) or b6 (B). The amount of captured virus was assessed by p24 ELISA. Each MAb was also competed against itself as a positive control. The controls (black bars) show the amounts of virus captured on b12- or b6-coated plates in the absence of GRFT. The bars represent the means and standard deviations (SD) of three different experiments.

Fig. 2.

CD4-IgG2 capture of HIV-1 was enhanced by GRFT. HIV-1 subtype B pseudovirus QH0692.42 (A) and the subtype C pseudovirus COT6.15 (B) were incubated with different concentrations of GRFT and added to a plate coated with CD4-IgG2. The amount of captured virus was assessed by p24 ELISA relative to an untreated control with no GRFT (black bars). CD4-IgG2 was also competed against itself as a positive control. The bars represent the means and SD of three different experiments.

Fig. 3.

CV-N competed with b12 and b6 for binding to HIV-1. The subtype B pseudovirus QH0692.42 and the subtype C pseudovirus COT6.15 were incubated with different concentrations of CV-N and then added to plates coated with either b12 (A and B) or b6 (C and D). The amount of captured virus was assessed by p24 ELISA relative to an untreated control with no CV-N (black bars). MAbs were also competed against themselves as positive controls. The bars represent the means and SD of three different experiments.

Fig. 4.

No enhancement of HIV-1 binding to F240-, 4E10-, and 3468L-coated plates by GRFT. The subtype B virus QH0692.42 was incubated with different concentrations of GRFT and then added to plates coated with the MAbs F240 (A), 4E10 (B), and 3468L (C). The amount of captured virus was assessed by p24 ELISA relative to an untreated control with no GRFT (black bars). Each antibody was also competed against itself as a positive control. The bars represent the means and SD of three different experiments.

Fig. 5.

Effects of the 386 glycosylation site on the HIV-1 inhibition curves of GRFT and b12. (A and B) HIV-1 QH0692.42 (A) and COT6-V295N/S448N (B) were neutralized in TZM-bl cells with GRFT and b12, in combination and alone. The shift in the inhibition curves of the two compounds combined relative to each compound alone are shown. (C and D) The same experiment as in panels A and B but using QH0692-N386Q and COT6-V295N/S448N/N386Q, respectively. The dashed lines indicate 50 and 80% inhibition. The graphs are representative of three different experiments.

Fig. 6.

GRFT inhibition of 17b MAb binding to the CD4i epitope. (A) The HIV-2 pseudovirus 7312A was first incubated with different concentrations of GRFT and then with 25 μg/ml sCD4 prior to capture on a 17b-coated plate. The well containing the immobilized 17b only (0 μg/ml of GRFT and sCD4) is the experimental control well. The amount of captured virus was measured by p24 ELISA. The bars represent the means and SD of three different experiments. (B) Same experiment as in panel A, except that 7312A was first incubated with 25 μg/ml of sCD4 before incubation with different concentrations of GRFT. (C) The HIV-2 pseudovirus 7312A was captured with CD4-IgG2 and then incubated with sCD4. This was followed by sequential addition of 17b, GRFT, and anti-GRFT antibody to the captured virus. The white bar (the well containing the virus and GRFT only) is the positive control, while the black bar (the well containing the virus only) is the negative control. The amount of GRFT bound to the virus was measured by measuring the optical density (O.D.) at 450 nM after the addition of horseradish peroxidase and the substrate. The bars represent the means and SD of three different experiments.

Fig. 7.

GRFT inhibited HIV-1 infection in U87-CCR5 and U87-CXCR4 cells. HIV-1 subtype C Du151, DS12, CM9, and RP1 were treated with different concentrations of GRFT before infection of U87-CCR5 (A) and U87-CXCR4 (B) cells. The data are shown as the averages plus standard deviations of three independent experiments. Untreated virus is shown in white (positive control). The IC₅₀s of GRFT are indicated above each graph.