Two N-linked glycosylation sites in the V2 and C2 regions of human immunodeficiency virus type 1 CRF01_AE envelope glycoprotein gp120 regulate viral neutralization susceptibility to the human monoclonal antibody specific for the CD4 binding domain

Abstract

A recombinant human monoclonal antibody, IgG1 b12 (b12), recognizes a conformational epitope on human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) gp120 that overlaps the CD4 binding domain. Although b12 is able to broadly neutralize HIV-1 subtype B, C, and D viruses, many HIV-1 CRF01_AE viruses are resistant to b12-mediated neutralization. In this report, we examined the molecular mechanisms underlying the low neutralization susceptibility of CRF01_AE viruses to b12, using recently established CRF01_AE Env recombinant viruses. Our results showed that two potential N-linked glycosylation (PNLG) sites in the V2 and C2 regions of Env gp120 played an important role in regulating the susceptibility of CRF01_AE Env to b12. The locations of these PNLG sites correspond to amino acid positions 186 and 197 in HXB2 Env gp120; thus, they are designated N186 and N197 in this study. Removal of N186 significantly conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones, 65CC4 and 107CC2, while the introduction of N186 reduced the b12 susceptibility of a susceptible CRF01_AE Env clone, 65CC1. In addition, removal of both N186 and N197 conferred the b12 susceptibility of 3 resistant CRF01_AE Env clones, 45PB1, 62PL1, and 101PL1, whereas the removal of either N186 or N197 was not sufficient to confer the b12 susceptibility of these CRF01_AE Env clones. Finally, removal of N197 conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones lacking N186, 55PL1 and 102CC2. Taken together, we propose that two PNLG sites, N186 and N197, in Env gp120 are important determinants of the b12 resistance of CRF01_AE viruses.

FIG. 1.

(A) Schematic illustration of variable (V) and conserved (C) regions of Env gp120 and gp41. Chimeric CRF01_AE Env containing partial fragments of 65CC1 and 65CC4, 65CC1N4C and 65CC4N1C, respectively, were constructed and subjected to neutralization tests. (B) N-terminal regions of gp120 contain the determinants of b12 resistance of 65CC4. Neutralization susceptibility of CRF01_AE Env recombinant viruses to b12 was evaluated as described in Materials and Methods. Results are expressed as percent neutralization, which was calculated by determining the reduction in viral infectivity in the presence of b12 compared to that in control experiments in the absence of b12. All data points represent the means and standard errors (error bars) of four independent experiments.

FIG. 2.

Comparison of the N-terminal regions of gp120 between 2 CRF01_AE Env clones, 65CC1 and 65CC4 (A), or among 5 subtype B and 2 CRF01_AE Env clones (B). Amino acid sequences of the N-terminal regions (before the XbaI recognition site) of Env gp120 were compared among subtype B strains, HXB2 (GenBank accession no. K03455), JR-FL (U63632), JR-CSF (M38429), 89.6 (U39362) and SF162 (M65024), as well as CRF01_AE Env, 65CC1 (EU743779), and 65CC4 (EU743780). These amino acid sequences were aligned using the ClustalW algorithm (44) with slight manual adjustment. The positions of gp120 variable (V) and conserved (C) regions are denoted in the figure. Dots denote amino acid identity, while dashes represent gaps introduced to optimize alignment. PNLG sites are shown by underlining. In panel B, the numbering of amino acid residues is based on the sequence of HXB2 Env and is shown beside the aligned sequence. Arrowheads indicate two PNLG sites, N186 and N187, detected in 65CC4 but not 65CC1, whereas asterisks indicate the amino acid difference in the V3 region in 65CC1 and 65CC4.

FIG. 3.

(A) Schematic illustration of the locations of amino acid substitutions introduced into CRF01_AE Env, 65CC1 and 65CC4. (B) Removal of the PNLG site, N186, confers b12 susceptibility of CRF01_AE Env, 65CC4, while introduction of N186 reduces b12 susceptibility of 65CC1. Neutralization susceptibility of CRF01_AE Env recombinant viruses to b12 was evaluated as described in Materials and Methods. Results are expressed as percent neutralization, as described in the legend to Fig. 1. All data points represent the means and standard errors (error bars) of three independent experiments.

FIG. 4.

(A and B) Removal of N186 significantly confers b12 susceptibility of CRF01_AE Env, 107CC2 (A), while removal of both N186 and N197 is required to confer b12 susceptibility of CRF01_AE Env, 45PB1, 62PL1, and 101PL1 (B). The susceptibility of CRF01_AE Env recombinant viruses, as well as pNL-envCT containing pNL4-3 Env, to b12-mediated neutralization was evaluated as described in Materials and Methods. Results are expressed as percent neutralization, as described in the legend to Fig. 1. All data points represent the means and standard errors (error bars) of three independent experiments.

FIG. 5.

Removal of N197 confers b12 susceptibility of CRF01_AE Env clones lacking N186, 55PL1 and 102CC2. The susceptibility of CRF01_AE Env recombinant viruses to b12-mediated neutralization was evaluated as described in Materials and Methods. Results are expressed as percent neutralization, as described in the legend to Fig. 1. All data points represent the means and standard errors (error bars) of three independent experiments.

FIG. 6.

No strict correlation is observed between the infectivity and susceptibility to b12 of CRF01_AE Env recombinant viruses. Viral infectivity of the N-linked glycosylation mutant virus lacking the PNLG site(s), N186Q, N197Q, or both N186 and N197 (N186Q/N197Q) was compared with that of the corresponding wild-type virus as described in Materials and Methods. Data are shown as fold difference in luciferase activity produced by the N-linked glycosylation mutant virus relative to luciferase activity produced by the wild-type virus. Results are presented as the means and standard errors (error bars) of three independent experiments. Differences among the viruses were analyzed by the paired t test and are reported when P < 0.05.

FIG. 7.

A model of the role of N-linked glycans at amino acid positions 186, 187, and 197 in the interaction between gp120 and b12. A structural model of glycosylated gp120 complexed with b12 was constructed on the basis of previously solved gp120 structures (PDB IDs 3DNL, 2NY7, and 2BF1) as described in Materials and Methods. The generated model has 3-fold rotational symmetry. Each complex of gp120 and b12 is represented by a different model, a ribbon model with distance measurements, a ribbon model with a surface, or a ribbon model with glycosylated gp120. The structures of gp120 and b12 are colored purple and green, respectively. In addition, the location of the V1/V2 stem is colored yellow. The unknown V1 and V2 structures and N-linked glycans are shown as a circle and triangles, respectively. Two bars indicate the distances in two different directions (55 and 67 Å) from the V1/V2 stem to b12 binding to the adjacent gp120 molecule.