The lectins griffithsin, cyanovirin-N and scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4(+) cells

Abstract

It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4(+) cells. The lectins griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN) inhibit HIV-1 infection by binding to mannose-rich glycans on gp120. We measured the ability of these lectins to inhibit both the HIV-1 binding to DC-SIGN and the DC-SIGN-mediated HIV-1 infection of CD4(+) cells. While GRFT, CV-N and SVN were moderately inhibitory to DC-SIGN binding, they potently inhibited DC-SIGN-transfer of HIV-1. The introduction of the 234 glycosylation site abolished HIV-1 sensitivity to lectin inhibition of binding to DC-SIGN and virus transfer to susceptible cells. However, the addition of the 295 glycosylation site increased the inhibition of transfer. Our data suggest that GRFT, CV-N and SVN can block two important stages of the sexual transmission of HIV-1, DC-SIGN binding and transfer, supporting their further development as microbicides.

Figure 1. DC-SIGN receptor expression on Raji/DC-SIGN cells

(A) Raji/DC-SIGN and Raji cells were stained with phycoerythrin (PE)-labeled mouse anti-human CD209 (DC-SIGN) and analyzed by flow cytometry. DC-SIGN expression is shown beneath the bar labeled M1. (B) HIV-1 subtype B, QH0692.42 and subtype C, COT6.15 were captured by Raji/DC-SIGN and Raji cells at 3 different cell concentrations. The amount of captured virus was measured by p24 ELISA. (C) Inhibition of HIV-1 binding to Raji/DC-SIGN cells by mouse anti-human CD209 antibody. The amount of captured virus was measured by p24 ELISA.

Figure 2. Preincubation of HIV-1 with GRFT, CV-N and SVN inhibited binding to the DC-SIGN receptor

HIV-1 was incubated with GRFT, CV-N and SVN prior to capture on Raji/DC-SIGN cells. Bound virus was lysed and the amount of captured p24 was measured by ELISA. Bars represent mean ± SD of three different experiments. Untreated controls are shown in black.

Figure 3. Effects of 234 and 295 glycans on GRFT, CV-N and SVN inhibition of HIV-1 binding to the DC-SIGN receptor

(A) CAAN5342.A2 and (B) CAP63.A9J mutants lacking 234N and 295N were captured with Raji/DC-SIGN cells in the presence of GRFT, CV-N and SVN. The amount of bound virus was measured by p24 ELISA. Bars represent mean ± SD of three different experiments. Untreated controls are shown in black. The highest percentage inhibition obtained with each lectin is show next to the graphs.

Figure 4. Inhibition of DC-SIGN mediated HIV-1 transfer by post and pre-DC-SIGN binding methods

(A) In the post-DC-SIGN binding method, HIV-1 subtype B QH0692.42 and subtype C COT9.6 were incubated with Raji/DC-SIGN cells prior to the addition of GRFT, CV-N and SVN and co-culture with TZM-bl cells. (B) In the pre-DC-SIGN binding method, the virus was first incubated with GRFT, CV-N and SVN prior to the addition of Raji/DC-SIGN cells and co-culture with TZM-bl cells. The inhibition of transfer for both methods was determined by measuring the RLU. Bars represent mean ± SD of three different experiments. Untreated controls are shown in black.

Figure 5. Comparison of IC₅₀ values between the post, pre and the combined method using TZM-bl cells

GRFT, CV-N and SVN inhibition of HIV-1 (A) COT9.6, (B) CAP63.A9J, (C) CAAN5342.A2 and (D) QH0692.42 transfer to TZM-bl cells was tested using the post, pre and combined methods. The IC₅₀ values obtained with each method were then compared. The values shown are average of three different experiments.

Figure 6. DC-SIGN-mediated HIV-1 transfer to PBMC is inhibited by GRFT, CV-N and SVN

HIV-1 subtype C infectious viruses SW7 and Du179 and subtype B QH0515 transfer to PBMC was inhibited with GRFT, CV-N and SVN using (A) the post and (B) pre-DC-SIGN binding methods. The inhibition of transfer was measured by p24 ELISA. Bars represent mean ± SD of three different experiments. Untreated controls are shown in black. Levels of p24 antigen in the absence of virus or lectins were below detection (not shown).

Figure 7. Comparison of IC₈₀ values between the post, pre and combined methods in PBMC

SW7 (A) and QH0515 (B) transfer to PBMC was inhibited with GRFT, CV-N and SVN using the post, pre and combined methods. This was followed by the comparison of IC₈₀ values obtained with each method. The values shown are average of three different experiments.