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. 2015 Oct 28;90(2):829-41.
doi: 10.1128/JVI.02321-15. Print 2016 Jan 15.

Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies against the Receptor and Coreceptor Binding Sites

Samantha Townsley  1 , Yun Li  2 , Yury Kozyrev  2 , Brad Cleveland  2 , Shiu-Lok Hu  3
Affiliations

Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies against the Receptor and Coreceptor Binding Sites

Samantha Townsley et al. J Virol. .

Abstract

HIV-1 establishes persistent infection in part due to its ability to evade host immune responses. Occlusion by glycans contributes to masking conserved sites that are targets for some broadly neutralizing antibodies (bNAbs). Previous work has shown that removal of a highly conserved potential N-linked glycan (PNLG) site at amino acid residue 197 (N7) on the surface antigen gp120 of HIV-1 increases neutralization sensitivity of the mutant virus to CD4 binding site (CD4bs)-directed antibodies compared to its wild-type (WT) counterpart. However, it is not clear if the role of the N7 glycan is conserved among diverse HIV-1 isolates and if other glycans in the conserved regions of HIV-1 Env display similar functions. In this work, we examined the role of PNLGs in the conserved region of HIV-1 Env, particularly the role of the N7 glycan in a panel of HIV-1 strains representing different clades, tissue origins, coreceptor usages, and neutralization sensitivities. We demonstrate that the absence of the N7 glycan increases the sensitivity of diverse HIV-1 isolates to CD4bs- and V3 loop-directed antibodies, indicating that the N7 glycan plays a conserved role masking these conserved epitopes. However, the effect of the N7 glycan on virus sensitivity to neutralizing antibodies directed against the V2 loop epitope is isolate dependent. These findings indicate that the N7 glycan plays an important and conserved role modulating the structure, stability, or accessibility of bNAb epitopes in the CD4bs and coreceptor binding region, thus representing a potential target for the design of immunogens and therapeutics.

Importance: N-linked glycans on the HIV-1 envelope protein have been postulated to contribute to viral escape from host immune responses. However, the role of specific glycans in the conserved regions of HIV-1 Env in modulating epitope recognition by broadly neutralizing antibodies has not been well defined. We show here that a single N-linked glycan plays a unique and conserved role among conserved glycans on HIV-1 gp120 in modulating the exposure or the stability of the receptor and coreceptor binding site without affecting the integrity of the Env in mediating viral infection or the ability of the mutant gp120 to bind to CD4. The observation that the antigenicity of the receptor and coreceptor binding sites can be modulated by a single glycan indicates that select glycan modification offers a potential strategy for the design of HIV-1 vaccine candidates.

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Figures

FIG 1
FIG 1
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).
FIG 2
FIG 2
V2 loop amino acid sequence of the panel of HIV-1 isolates. The amino acid sequence of the V2 loop for each Env (25, 28, 29, 31, 32, 99–101) was aligned to the HXB2 reference sequence (102) 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.
FIG 3
FIG 3
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.
FIG 4
FIG 4
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. [21]). 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.
FIG 5
FIG 5
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 (61). 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).
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