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Cited by 115HIV Research and TreatmentGlycosylation and Glycoproteins ResearchCarbohydrate Chemistry and Synthesis

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A systematic study of the N-glycosylation sites of HIV-1 envelope protein on infectivity and antibody-mediated neutralization

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Wang, Wenbo, Nie, Jianhui, Prochnow, Courtney, Truong, Carolyn, Jia, Zheng, Wang, Suting, Chen, Xiaojiang S, Wang, Youchun·2013·Retrovirology
DOI 10.1186/1742-4690-10-14PMC PMC3648360
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Abstract

BACKGROUND: Glycans on the human immunodeficiency virus (HIV) envelope glycoprotein (Env) play an important role in viral infection and evasion of neutralization by antibodies. In this study, all 25 potential N-linked glycosylation sites (PNGS) on the HIV-1 CRF07_BC Env, FE, were mutated individually to study the effect of their removal on viral infectivity, virion production, and antibody-mediated neutralization. RESULTS: Removal of specific N-glycosylation sites has a significant effect on viral infectivity and antibody-mediated neutralization phenotype. Six of these glycosylation mutants located on the V1/V2 and C1/C2 domains lost infectivity. PNGS mutations located on V4/C4/V5 (except N392 on V4), were shown to increase viral infectivity. Furthermore, FE is much more dependent on specific glycans than clade B Env YU-2. On neutralization effect, PNGS mutations at N197 (C2), N301 (V3), N442 (C4) and N625 (gp41) rendered the virus more susceptible to neutralization by the monoclonal antibodies (MAbs) that recognize the CD4 binding site or gp41. Generally, mutations on V4/V5 loops, C2/C3/C4 regions and gp41 reduced the neutralization sensitivity to PG16. However, mutation of N289 (C2) made the virus more sensitive to both PG9 and PG16. Furthermore, we showed that mutations at N142 (V1), N355 (C3) and N463 (V5) conferred resistance to neutralization by anti-gp41 MAbs. We used the available structural information of HIV Env and homology modeling to provide a structural basis for the observed biological effects of these mutations. CONCLUSIONS: This report provides the first systematic experimental account of the biological role of the entire PNGS on an HIV-1 Env, which should provide valuable insights for understanding the function of Env in HIV infection cycle and for developing future anti-HIV strategies.

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Figure 1 Infectivity assay of wt HIV strains and the PNGS mutants. a) Comparison of the infectivity of wt HIV-1 strains: FE and four other strains. b) Infectivity assay result of all FE PNGS mutants. c) Comparison of relative infectivity of some of the PNGS mutants in FE, Sc19-15 and YU-2 isolates. Data are shown as relative luminescence units (RLU) in a logarithmic scale. Relative infectivity was calculated by dividing the Log10 (RLU of mutant) by Log10 (RLU of wt). The data represent the means of three independent experiments, and the error bars indicate the standard deviations from the means. The cut off value of the infectivity assay is 4.0. #, glycan deletion mutant that could not support virus entry.
Figure 1 Infectivity assay of wt HIV strains and the PNGS mutants. a) Comparison of the infectivity of wt HIV-1 strains: FE and four other strains. b) Infectivity assay result of all FE PNGS mutants. c) Comparison of relative infectivity of some of the PNGS mutants in FE, Sc19-15 and YU-2 isolates. Data are shown as relative luminescence units (RLU) in a logarithmic scale. Relative infectivity was calculated by dividing the Log10 (RLU of mutant) by Log10 (RLU of wt). The data represent the means of three independent experiments, and the error bars indicate the standard deviations from the means. The cut off value of the infectivity assay is 4.0. #, glycan deletion mutant that could not support virus entry.CC BY · PubMed Central
Figure 2 Structure models of FE Env, gp120 and gp41. a) Two views of the model of the FE gp120 monomer with grafted V1/V2 domain (see Methods). The gp120 monomer consists of four domains: the inner domain (light blue), the outer domain (light pink), the bridging sheet (orange), and the V1/V2 loop (dark blue). The 4 beta stranded bridging sheet forms upon CD4 binding near the CD4 binding loop (yellow). The V3 loop (light green) extends from the outer domain. Mutated PNGS that increase or decrease viral neutralization by any of the MAbs are shown as red sticks, and sites with negligible effects are shown as blue sticks. b) Two views of the model of the FE gp41 six helix bundle (see Methods). The three alpha helices in the middle are shown in cyan, and the three helices on the outside are in green. Mutated PNGS N625 and N637 are shown as red sticks. Aromatic residues W678, W680, and Y681 (yellow) from the MPRE region extend from the tip of each monomer and are proposed to insert into the viral membrane (only one set of residues shown).
Figure 2 Structure models of FE Env, gp120 and gp41. a) Two views of the model of the FE gp120 monomer with grafted V1/V2 domain (see Methods). The gp120 monomer consists of four domains: the inner domain (light blue), the outer domain (light pink), the bridging sheet (orange), and the V1/V2 loop (dark blue). The 4 beta stranded bridging sheet forms upon CD4 binding near the CD4 binding loop (yellow). The V3 loop (light green) extends from the outer domain. Mutated PNGS that increase or decrease viral neutralization by any of the MAbs are shown as red sticks, and sites with negligible effects are shown as blue sticks. b) Two views of the model of the FE gp41 six helix bundle (see Methods). The three alpha helices in the middle are shown in cyan, and the three helices on the outside are in green. Mutated PNGS N625 and N637 are shown as red sticks. Aromatic residues W678, W680, and Y681 (yellow) from the MPRE region extend from the tip of each monomer and are proposed to insert into the viral membrane (only one set of residues shown).CC BY · PubMed Central
Figure 3 Viral relative infectivity and virion incorporation of mutant gp120. Gp120 incorporation data are shown as the percentage of the gp120/p24 ratio of the wt FE pseudovirus, with the wt gp120 incorporation set to 1.0. Relative infectivity was calculated by dividing the Log10 (RLU of mutant) by Log10 (RLU of wt). The data represent the means of three independent experiments, and the error bars indicate the standard deviations from the means. P values were calculated by using One-way analysis of variance (Tukey’s multiple comparison Test, SPSS 10.0). The difference of the mean infectivity and gp120 incorporation between wt and mutant is considered to be significant when a p value is <0.05, which is indicated with an asterisk in the figure.
Figure 3 Viral relative infectivity and virion incorporation of mutant gp120. Gp120 incorporation data are shown as the percentage of the gp120/p24 ratio of the wt FE pseudovirus, with the wt gp120 incorporation set to 1.0. Relative infectivity was calculated by dividing the Log10 (RLU of mutant) by Log10 (RLU of wt). The data represent the means of three independent experiments, and the error bars indicate the standard deviations from the means. P values were calculated by using One-way analysis of variance (Tukey’s multiple comparison Test, SPSS 10.0). The difference of the mean infectivity and gp120 incorporation between wt and mutant is considered to be significant when a p value is <0.05, which is indicated with an asterisk in the figure.CC BY · PubMed Central
Figure 4 Neutralization susceptibility of WT FE and PNGS mutants by several known neutralizing MAbs. All data points represent the means and the error bars indicate the standard deviations from the means that were obtained from three independent experiments. Neutralization assays of the pseudovirions produced from WT and mutants were described in Materials and Methods.
Figure 4 Neutralization susceptibility of WT FE and PNGS mutants by several known neutralizing MAbs. All data points represent the means and the error bars indicate the standard deviations from the means that were obtained from three independent experiments. Neutralization assays of the pseudovirions produced from WT and mutants were described in Materials and Methods.CC BY · PubMed Central
Figure 5 Modeled FE gp120 trimers. a) Model of unliganded FE gp120 trimer containing the truncated V1/V2 stem loop in a “closed” conformation (Upper and lower panels). Top and front views showing the closed conformation of an HIV-1 FE gp120 trimer. We were unable to model the recently solved complete V1/V2 domain onto the unliganded trimer model due to steric clashes; however, a portion of the truncated V1/V2 domain, the V1/V2 stem loop (dark blue), can be seen in a “closed” conformation near the center of the trimer. More structural information is needed regarding the unliganded full-length gp120 trimer to determine precisely where the complete V1/V2 domain is positioned in this conformation; however, owing to its flexibility, the V1/V2 domain will likely also be positioned near the center of the trimer. This is also consistent with cryo-electron tomography studies [26,28]. b) Model of CD4 bound FE gp120 trimer containing the complete V1/V2 domain in an “open” conformation. Top and front views showing the open conformation of the trimer upon binding the CD4 receptor. We were able to graft the complete V1/V2 domain (dark blue) onto the gp120 core. Upon CD4 binding, the V1/V2 domain rotates outwardly in an “open” orientation, consistent with cryo-electron tomography studies [26,28].
Figure 5 Modeled FE gp120 trimers. a) Model of unliganded FE gp120 trimer containing the truncated V1/V2 stem loop in a “closed” conformation (Upper and lower panels). Top and front views showing the closed conformation of an HIV-1 FE gp120 trimer. We were unable to model the recently solved complete V1/V2 domain onto the unliganded trimer model due to steric clashes; however, a portion of the truncated V1/V2 domain, the V1/V2 stem loop (dark blue), can be seen in a “closed” conformation near the center of the trimer. More structural information is needed regarding the unliganded full-length gp120 trimer to determine precisely where the complete V1/V2 domain is positioned in this conformation; however, owing to its flexibility, the V1/V2 domain will likely also be positioned near the center of the trimer. This is also consistent with cryo-electron tomography studies [26,28]. b) Model of CD4 bound FE gp120 trimer containing the complete V1/V2 domain in an “open” conformation. Top and front views showing the open conformation of the trimer upon binding the CD4 receptor. We were able to graft the complete V1/V2 domain (dark blue) onto the gp120 core. Upon CD4 binding, the V1/V2 domain rotates outwardly in an “open” orientation, consistent with cryo-electron tomography studies [26,28].CC BY · PubMed Central

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Research works printed but not linked: 1

  1. Human Retroviruses and AIDS 1989: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Edited by: Myers GRA, Josephs SF, Smith TF, Wong-Staal F, Berzofsky JA. 1989, Los Alamos, NM: Theoretical Biology and Biophysics Group, Los Alamos National Laboratory

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Metadata

Authors
Wang, W., Nie, J., Prochnow, C., Truong, C., Jia, Z., Wang, S., Chen, X. S., & Wang, Y.
Affiliations
  • National Institutes for Food and Drug Control (CN)
  • University of Southern California (US)
Corresponding author
Wang, Youchun
ORCID
Venue
Retrovirology
Publisher
Springer Science and Business Media LLC
Published
2013-02-06
Volume
10
Issue
1
Pages
14-14
ISSN
1742-4690 (ISSN-L)
DOI
10.1186/1742-4690-10-14
PMC
PMC3648360
OpenAlex
W2137366946
Cited by
114
References
64
Citations by year
2026: 3 · 2025: 10 · 2024: 6 · 2023: 4 · 2022: 5 · 2021: 16 · 2020: 8 · 2019: 9 · 2018: 8 · 2017: 11 · 2016: 8 · 2015: 13 · 2014: 8 · 2013: 4
FWCI
5.05
Type
journal article
Language
en
Open access
Licence
Locations
  • Retrovirology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (GM087986)
  • National Institute of General Medical Sciences (GM087986, R01GM087986)
Topics
  • Life Sciences › Immunology and Microbiology › Virology › HIV Research and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Physical Sciences › Chemistry › Organic Chemistry › Carbohydrate Chemistry and Synthesis
Keywords
HIV-1 envelope glycoprotein, N-glycosylation sites, viral infectivity, antibody neutralization, CRF07_BC, V1V2 loop, gp41, CD4 binding site, monoclonal antibodies, PG9, homology modeling, HIV-1 clade B
MeSH terms
Glycosylation, Humans, Neutralization Tests, Virus Replication, HIV Antibodies, HIV-1, env Gene Products, Human Immunodeficiency Virus, Antibodies, Neutralizing

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