Wenbo Wang1, Brett Zirkle, Jianhui Nie, Jian Ma, Kai Gao, Xiaojiang S Chen, Weijing Huang, Wei Kong, Youchun Wang. 1. *College of Life Science, Jilin University, Changchun, China; †Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Division of Monoclonal Antibody, National Institutes for Food and Drug Control (NIFDC), Beijing, China; ‡Department of Molecular and Computational Biology/Chemistry Department/Norris Cancer Center, University of Southern California, Los Angeles, CA; and §Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, National Institutes for Food and Drug Control (NIFDC), Beijing, China.
Abstract
BACKGROUND: HIV-1 gp120/gp41 is heavily modified by n-linked carbohydrates that play important roles either in correct folding or in shielding vulnerable viral protein surfaces from antibody recognition. METHODS: In our previous work, 25 potential N-linked glycosylation sites (PNGS) of a CRF07_BC isolate of HIV-1 were individually mutated, and the resulting effects on infectivity and antibody-mediated neutralization were evaluated. To further understand the functional role of these PNGS, we generated double and multiple mutants from selected individual PNGS mutants. The effects were then evaluated by examining infectivity and sensitivity to antibody-mediated neutralization by neutralizing monoclonal antibodies (nMAbs) and serum antibodies from HIV-1 positive donors. RESULTS: Infectivity results showed that, among the 12 combined PNGS mutants, only 197M.1 (N197D/N301Q) lost infectivity completely, whereas all others (except for 197M.6) showed reduced viral infectivity. In terms of neutralization sensitivity to known nMAbs, we found that adding N463Q mutation to all the gp120 mutants containing N197D significantly increased neutralization sensitivity to VRC01 and VRC03, suggesting N197 and N463 have a strong synergistic effect in regulating the neutralizing sensitivity of HIV-1 to the anti-CD4bs nMAbs VRC01/VRC03. Structural analysis based on the available structures of gp120 alone and in complex with CD4 and various nMAbs elucidates a molecular rationale for this experimental observation. CONCLUSIONS: The data indicate that N463 plays an important role in regulating the CD4bs MAbs VRC01/VRC03 sensitivity in the genetic background of N197D mutation of gp120, which should provide valuable information for a better understanding of the interplay between HIV-1 and VRC01/03.
BACKGROUND:HIV-1gp120/gp41 is heavily modified by n-linked carbohydrates that play important roles either in correct folding or in shielding vulnerable viral protein surfaces from antibody recognition. METHODS: In our previous work, 25 potential N-linked glycosylation sites (PNGS) of a CRF07_BC isolate of HIV-1 were individually mutated, and the resulting effects on infectivity and antibody-mediated neutralization were evaluated. To further understand the functional role of these PNGS, we generated double and multiple mutants from selected individual PNGS mutants. The effects were then evaluated by examining infectivity and sensitivity to antibody-mediated neutralization by neutralizing monoclonal antibodies (nMAbs) and serum antibodies from HIV-1 positive donors. RESULTS: Infectivity results showed that, among the 12 combined PNGS mutants, only 197M.1 (N197D/N301Q) lost infectivity completely, whereas all others (except for 197M.6) showed reduced viral infectivity. In terms of neutralization sensitivity to known nMAbs, we found that adding N463Q mutation to all the gp120 mutants containing N197D significantly increased neutralization sensitivity to VRC01 and VRC03, suggesting N197 and N463 have a strong synergistic effect in regulating the neutralizing sensitivity of HIV-1 to the anti-CD4bsnMAbs VRC01/VRC03. Structural analysis based on the available structures of gp120 alone and in complex with CD4 and various nMAbs elucidates a molecular rationale for this experimental observation. CONCLUSIONS: The data indicate that N463 plays an important role in regulating the CD4bs MAbs VRC01/VRC03 sensitivity in the genetic background of N197D mutation of gp120, which should provide valuable information for a better understanding of the interplay between HIV-1 and VRC01/03.
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