Mannose-rich glycosylation patterns on HIV-1 subtype C gp120 and sensitivity to the lectins, Griffithsin, Cyanovirin-N and Scytovirin

Abstract

Griffithsin (GRFT), Cyanovirin-N (CV-N) and Scytovirin (SVN) are lectins that inhibit HIV-1 infection by binding to multiple mannose-rich glycans on the HIV-1 envelope glycoproteins (Env). Here we show that these lectins neutralize subtype C primary virus isolates in addition to Env-pseudotyped viruses obtained from plasma and cervical vaginal lavages. Among 15 subtype C pseudoviruses, the median IC(50) values were 0.4, 1.8 and 20.1nM for GRFT, CV-N and SVN, respectively, similar to what was found for subtype B and A. Analysis of Env sequences suggested that concomitant lack of glycans at positions 234 and 295 resulted in natural resistance to these compounds, which was confirmed by site-directed mutagenesis. Furthermore, the binding sites for these lectins overlapped that of the 2G12 monoclonal antibody epitope, which is generally absent on subtype C Env. This data support further research on these lectins as potential microbicides in the context of HIV-1 subtype C infection.

Figure 1. GRFT, CV-N, and SVN inhibit HIV-1 subtype C infection of PBMC

Three primary isolates, Du151 (A), COT9 (B) and Du179 (C) were treated with increasing concentrations of GRFT, CV-N and SVN before infection of PBMC. Data are shown as the average plus standard deviations of three independent experiments. Untreated virus is shown in black (positive control). The IC₈₀ values of the lectins areindicated next to each graph.

Figure 2. GRFT, CV-N, and SVN inhibit HIV-1 infection in the TZM-bl assay

Neutralization of HIV-1 subtype C, B and A pseudoviruses by GRFT (A), CV-N (B), and SVN (C). HIV-1 subtype B and A pseudoviruses were used for comparison to subtype C. Each virus was tested at least three times. Pseudoviruses are ranked by GRFT sensitivity.

Figure 3. GRFT, CV-N, and SVN inhibit HIV-1 isolates from CVL

Sensitivity of 4 HIV-1 subtype C pseudoviruses containing functional envelope genes amplified from cervico-vaginal lavages against GRFT (A), CV-N (B) and SVN (C) are shown.

Figure 4. Glycans at positions 234 and 295 increase HIV-1 sensitivity to GRFT, CV-N and SVN

Glycosylation sites at 234 and/or 295 were introduced in CAP206.08J, CAP63.A9J and CAAN5342.A2 by site-directed mutagenesis. Mutant viruses were tested in neutralization assays against GRFT (A), CV-N (B) and SVN (C) in JC53bl-13 cells. Each virus was tested at least three times.

Figure 5. GRFT, CV-N, and SVN compete with the 2G12 mAb for binding to HIV-1

Pseudoviruses QH0692.42 (A), COT6.15-V295N/S448N (B) and COT9.6-V295N/K442N (C), the latter two with a reconstituted 2G12 epitope, were incubated with GRFT, CV-N and SVN prior to capture with the 2G12 mAb. 2G12 was competed against itself as the experimental control. Experiments were done at least 3 times.