Mutational pathways, resistance profile, and side effects of cyanovirin relative to human immunodeficiency virus type 1 strains with N-glycan deletions in their gp120 envelopes

Abstract

Limited data are available on the genotypic and phenotypic resistance profile of the alpha-(1-2)mannose oligomer-specific prokaryotic lectin cyanovirin (CV-N). Therefore, a more systematic investigation was carried out to obtain a better view of the interaction between CV-N and human immunodeficiency virus type 1 (HIV-1) gp120. When HIV-1-infected CEM cell cultures were exposed to CV-N in a dose-escalating manner, a total of eight different amino acid mutations exclusively located at N-glycosylation sites in the envelope surface gp120 were observed. Six of the eight mutations resulted in the deletion of high-mannose type N-glycans (i.e., at amino acid positions 230, 332, 339, 386, 392, and 448). Two mutations (i.e., at position 136 and 160) deleted a complex type N-glycan in the variable V1/V2 domain of gp120. The level of phenotypic resistance of the mutated virus strains against CV-N generally correlated with the number of glycan deletions in gp120, although deletion of the glycans at N-230, N-392, and N-448 generally afforded a more pronounced CV-N resistance than other N-glycan deletions. However, the extent of the decrease of antiviral activity of CV-N against the mutated virus strains was markedly less pronounced than observed for alpha(1-3)- and alpha(1-6)-mannose-specific plant lectins Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA), which points to the existence of a higher genetic barrier for CV-N. This is in agreement with a more consistent suppression of a wider variety of HIV-1 clades by CV-N than by HHA and GNA. Whereas the antiviral and in vitro antiproliferative activity of CV-N can be efficiently reversed by mannan, the pronounced mitogenic activity of CV-N on peripheral blood mononuclear cells was unaffected by mannan, indicating that some of the observed side effects of CV-N are unrelated to its carbohydrate specificity/activity.

FIG. 1.

Resistance selection in HIV-1(III_B)-infected (A and B) or HIV-1 (NL-4.3)-infected (C) CEM cell cultures in the presence of escalating concentrations of CV-N. Every 4 to 5 days, the cell cultures were subcultivated by adding 100 or 200 μl of the virus-infected cell suspension to 900 or 800 μl fresh culture medium, respectively.

FIG. 2.

HIV-1 p24 production in PBMC cultures that are infected by different HIV-1 strains in the presence of various CV-N concentrations. The NL-4.3 strain is X4-tropic; the Ba-L, ETH2220, ID 12, BZ163, and UG273 strains are R5-tropic. The p24 levels in the PBMC supernatants are expressed as percentages of p24 production in the untreated virus-infected control cultures.

FIG. 3.

Ribbon representation of the HIV-1 gp120 envelope glycoprotein (blue) bound to CD4 receptor (pink), as found in a composite of X-ray crystal structures with Protein Data Bank identification codes 1GC1 (19, 32) and 2B4C (18). Sugar moieties are displayed as sticks, with C atoms colored white for N-acetyl-d-glucosamine and green for fucose. The Cbeta atoms of the glycosylated N residues that are mutated in the presence of CV-N are displayed as spheres and have been labeled. The PyMOL molecular graphics system (http://www.pymol.org; DeLano Scientific LLC, San Carlos, CA) was used for visualization and picture creation. Clustering was done manually on the basis of proximity of the glycosylated sites. Residues 127 to 192 making up loops V1 and V2 are absent in the crystal structures of gp120. Therefore, the approximate positions of the N-136 and N-160 residues are indicated by arrows.

FIG. 4.

Effect of 2.5 mg/ml mannan on the anti-HIV-1 activity of CV-N in CEM cell cultures (A) and on cell viability in CEM (B) and PBMC (C) cell cultures. CPE, cytopathic effect.

FIG. 5.

Mitogenic activity of CV-N in PBMC. [³H]thymidine ([³H]dThd) incorporation in TCA-insoluble cell material was quantified after a 3-day incubation period of PBMC with CV-N or PHA, followed by 20 h of exposure to [³H]thymidine. dpm, disintegrations per minute.

FIG. 6.

Effect of CV-N on the expression of three activation markers in PBMC. (A) CD25⁺ CD4⁺ cell population; (B) CD69⁺ CD4⁺ cell population; (C) HLA-DR⁺ CD4⁺ cell population.