PMC

Infectivities of viruses pseudotyped with WT or mutant N7 Env. The relative infectivities of viruses pseudotyped with WT or N7 mutant Env from ADA, B33, or PVO.4 were measured as the quantity of p24 (in picograms) normalized to the infectivity (TCID₅₀) of the respective viruses. Solid bars indicate viruses pseudotyped with the N7-glycosylated form of Env and hatched bars the N7-deglycosylated forms. Results for all viruses are shown, with the average and standard deviation obtained from three independent experiments performed in duplicates.

V2 loop amino acid sequence of the panel of HIV-1 isolates. The amino acid sequence of the V2 loop for each Env (, , , , , ) was aligned to the HXB2 reference sequence () using ClustalW. Numbering is based on the HXB2 amino acid sequence. Identical amino acid residues are indicated by dots. Dashes indicate gaps. PNLG sites in each isolate are indicated by a red letter N. The N7 PNLG site is at amino acid position 197.

Neutralization sensitivity of viruses pseudotyped with WT 89.6 Env or PNLG mutants in the conserved regions of Env. Mutants were designated by the position of the asparagine residue of the PNLG sequon. The N197 mutant is abbreviated as N7. Neutralization assay was performed using sCD4, CD4-IgG, broadly neutralizing monoclonal antibodies, or pooled HIV-positive serum at concentrations or serum dilutions as indicated. Neutralization was quantified as the percent inhibition of viral infectivity measured by RLU expressed in TZM-bl indicator cells. The dotted line shows the concentration or reciprocal serum dilutions that resulted in 50% inhibition of viral infectivity (IC₅₀).

CD4 dependence of infection by WT and N7 glycan mutants. The infectivities of WT and N7 mutants were compared in cells expressing coreceptor CCR5 or CXCR4, with or without coexpression of CD4. The infectivity of R5 tropic viruses was determined in NP2 cells expressing CCR5. The infectivity of 89.6 was determined using U87 cells expressing CXCR4 (adapted from the method of Li et al. []). The inoculum used for each virus was normalized by its infectivity in isogenic cells expressing the CD4 receptor (200,000 RLU, measured in NP2-CCR5-CD4 or U87-CXCR4-CD4 cells). Solid bars indicate infectivity of viruses with the N7 PNLG present, hatched bars indicate N197Q mutants, and the striped bar indicates ADA N197S mutant virus. The results in all panels represent the averages from at least two independent experiments performed in duplicates.

Effect of N7 glycan on the binding of a monoclonal antibody recognizing a conformation-dependent epitope in the V2 loop. Binding of monoclonal antibody 697-30D to the WT or N7 mutant forms of SF162 (A) or JR-FL (B) Env was measured by antigen capture assays. Three different forms of Env were tested: (i) whole virions pseudotyped with the indicated Env, (ii) pseudovirions lysed with Triton X-100, or (iii) gp120 expressed in recombinant vaccinia virus-infected cells (). Binding to 697-30D was normalized to binding obtained with HIV-positive serum, and values obtained with the N7-deglycosylated Env (red bars) were expressed as a percentage of the values obtained with the N7-glycosylated Env (blue bars). The results represent the averages and standard deviations obtained from three individual experiments performed in duplicates. P values were calculated by the Wilcoxon matched-pairs signed-rank test (**, P ≤ 0.01).