Mechanisms of HIV-1 subtype C resistance to GRFT, CV-N and SVN

Abstract

We examined the ability of HIV-1 subtype C to develop resistance to the inhibitory lectins, griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN), which bind multiple mannose-rich glycans on gp120. Four primary HIV-1 strains cultured under escalating concentrations of these lectins became increasingly resistant tolerating 2 to 12 times their 50% inhibitory concentrations. Sequence analysis of gp120 showed that most had deletions of 1 to 5 mannose-rich glycans. Glycosylation sites at positions 230, 234, 241, 289 located in the C2 region and 339, 392 and 448 in the C3-C4 region were affected. Furthermore, deletions and insertions of up to 5 amino acids in the V4 region were observed in 3 of the 4 isolates. These data suggest that loss of glycosylation sites on gp120 as well as rearrangement of glycans in V4 are mechanisms involved in HIV-1 subtype C escape from GRFT, CV-N and SVN.

Keywords: Cyanovirin-N; Entry inhibitor; Glycans; Griffithsin; HIV subtype C; Microbicide; Resistance; Scytovirin; Single genome amplification.

Figure 1. In vitro generation of GRFT, CV-N and SVN resistant viruses

Primary HIV-1 subtype C isolates Du179 (A), Du151 (B), Du422 (C) and COT9 (D) were cultured in PBMC under escalating concentrations of GRFT, CV-N and SVN. The concentration of each lectin was gradually increased or reduced depending on the viral growth compared to the control cultures (containing no lectin) as determined by p24 antigen ELISA. The arrows indicate the time-point that the supernatant was collected for analysis.

Figure 2. Lectin-selected viruses showed a decreased sensitivity to GRFT, CV-N and SVN

Resistant primary HIV-1 subtype C isolates Du179 (A), Du151 (B), Du422 (C) and COT9 (D) were tested against GRFT, CV-N and SVN in a PBMC neutralization assay. The neutralization of HIV-1 infection was measured by p24 ELISA and the IC₈₀ of the resistant virus and the corresponding wild-type were determined by linear regression. Bars represent standard deviation of three independent experiments.

Figure 3. Cross-resistance between GRFT, CV-N and SVN

Du179 virus selected by GRFT (A), CV-N (B) and SVN (C) were tested against all three lectins in a PBMC assay. HIV-1 neutralization was measured by p24 ELISA and the IC₈₀ of the resistant virus (grey bar) and the corresponding wild-type (white bar) were determined by linear regression. Bars represent standard deviation of three independent experiments.

Figure 4. Mannose-rich glycosylation sites deleted in GRFT, CV-N and SVN selected viruses

The X-axis shows the positions of mannose-rich glycans deleted in the four isolates under lectin selective pressure. The positions of glycans are numbered according to the HxB2 virus (Leonard et al., 1990) and were identified by sequence analysis. The Y-axis shows the number of resistant viruses (out of 4) that had the deletion.

Figure 5. Changes in gp120 associated with lectin selection

Position of mannose-rich glycans and amino acid sequences for Du179 (A), Du151 (B), Du422 (C) and COT9 (D) following selection by GRFT, CV-N and SVN. Glycan deletions are shown in red and the addition in green. Symbols indicate changes in response to GRFT (*), CV-N (3) and SVN (3). Deletions and insertions of amino acid sequences in V4 are shown.

Figure 6. Amino acid sequence of isolated GRFT, CV-N and SVN resistant Du179 clones

GRFT (A), CV-N (B) and SVN (C) resistant Du179 clones gp120 sequence isolated by single genome amplification were aligned with their respective population sequences. The grey shading shows the presence of a potential mannose-rich glycosylation site, yellow shading indicates the site deletion while the red box shows the tip of the V3 loop (McCaffrey et al., 2004). Note that the glycan at position 393 is labeled as 392 in the text for comparison with other viruses.

Figure 6. Amino acid sequence of isolated GRFT, CV-N and SVN resistant Du179 clones

GRFT (A), CV-N (B) and SVN (C) resistant Du179 clones gp120 sequence isolated by single genome amplification were aligned with their respective population sequences. The grey shading shows the presence of a potential mannose-rich glycosylation site, yellow shading indicates the site deletion while the red box shows the tip of the V3 loop (McCaffrey et al., 2004). Note that the glycan at position 393 is labeled as 392 in the text for comparison with other viruses.