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The carbohydrate at asparagine 386 on HIV-1 gp120 is not essential for protein folding and function but is involved in immune evasion.

Sanders RW, van Anken E, Nabatov AA, Liscaljet IM, Bontjer I, Eggink D, Melchers M, Busser E, Dankers MM, Groot F, Braakman I, Berkhout B, Paxton WA - Retrovirology (2008)

Bottom Line: In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12.Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent.Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Experimental Virology, Dept, Medical Microbiology, Center of Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands. r.w.sanders@amc.uva.nl

ABSTRACT

Background: The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for approximately 50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain.

Results: The 386 carbohydrate was not essential for folding of wt gp120. However, its removal improved folding of a gp120 variant lacking the 385-418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies.

Conclusion: The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.

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Inhibition by inhibitors of receptor interactions. A. Locations of mutations and reversions on the structure of gp120 relative to the receptors binding sites. In this orientation the cell membrane would be on top, the viral membrane below and allows a direct view on the CD4BS. The lower panels show a 90° rotated view, a view from the target membrane. Residue 418 is indicated in yellow, and residues 386 and 433 are indicated in red. The GlcNac2Man3 core pentose of the carbohydrate attached to N386 is indicated in cyan (modeled onto gp120 by Dr. Peter Kwong). The residues important for the interaction with CD4 and the coreceptor are indicated in blue and green, respectively. B. Inhibition of virus variants with sCD4 and AMD3100 on CD4+ enriched lymphocytes. Inhibition curves are depicted for each of the viruses in limiting dilutions of either sCD4 (left panel) or the CXCR4 inhibitor AMD3100 (right panel).
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Figure 4: Inhibition by inhibitors of receptor interactions. A. Locations of mutations and reversions on the structure of gp120 relative to the receptors binding sites. In this orientation the cell membrane would be on top, the viral membrane below and allows a direct view on the CD4BS. The lower panels show a 90° rotated view, a view from the target membrane. Residue 418 is indicated in yellow, and residues 386 and 433 are indicated in red. The GlcNac2Man3 core pentose of the carbohydrate attached to N386 is indicated in cyan (modeled onto gp120 by Dr. Peter Kwong). The residues important for the interaction with CD4 and the coreceptor are indicated in blue and green, respectively. B. Inhibition of virus variants with sCD4 and AMD3100 on CD4+ enriched lymphocytes. Inhibition curves are depicted for each of the viruses in limiting dilutions of either sCD4 (left panel) or the CXCR4 inhibitor AMD3100 (right panel).

Mentions: To assess the conformation and function of the mutant and revertant Env proteins on virus particles, we studied the sensitivity of the revertant viruses to inhibitors of viral entry in a PBMC-based neutralization assay (fig. 4). The location of the binding sites of the receptor, CD4, and the CXCR4 coreceptor for HIV-1LAI in relation to the positions of the mutations and reversions in our viruses is given in fig. 4A. Soluble CD4 (sCD4) was used to assess the interaction of these viruses with CD4. The wt virus was neutralized by sCD4 with an IC50 of ~16 μg/ml. The various viruses (except mut, which did not replicate in PBMCs), were neutralized efficiently at lower concentrations of sCD4 (IC50 of ~1–8 μg/ml) suggesting that the affinity for CD4 is increased in these variants. R1b was the most sensitive to neutralization by sCD4 (IC50 of ~1.0 μg/ml).


The carbohydrate at asparagine 386 on HIV-1 gp120 is not essential for protein folding and function but is involved in immune evasion.

Sanders RW, van Anken E, Nabatov AA, Liscaljet IM, Bontjer I, Eggink D, Melchers M, Busser E, Dankers MM, Groot F, Braakman I, Berkhout B, Paxton WA - Retrovirology (2008)

Inhibition by inhibitors of receptor interactions. A. Locations of mutations and reversions on the structure of gp120 relative to the receptors binding sites. In this orientation the cell membrane would be on top, the viral membrane below and allows a direct view on the CD4BS. The lower panels show a 90° rotated view, a view from the target membrane. Residue 418 is indicated in yellow, and residues 386 and 433 are indicated in red. The GlcNac2Man3 core pentose of the carbohydrate attached to N386 is indicated in cyan (modeled onto gp120 by Dr. Peter Kwong). The residues important for the interaction with CD4 and the coreceptor are indicated in blue and green, respectively. B. Inhibition of virus variants with sCD4 and AMD3100 on CD4+ enriched lymphocytes. Inhibition curves are depicted for each of the viruses in limiting dilutions of either sCD4 (left panel) or the CXCR4 inhibitor AMD3100 (right panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2262092&req=5

Figure 4: Inhibition by inhibitors of receptor interactions. A. Locations of mutations and reversions on the structure of gp120 relative to the receptors binding sites. In this orientation the cell membrane would be on top, the viral membrane below and allows a direct view on the CD4BS. The lower panels show a 90° rotated view, a view from the target membrane. Residue 418 is indicated in yellow, and residues 386 and 433 are indicated in red. The GlcNac2Man3 core pentose of the carbohydrate attached to N386 is indicated in cyan (modeled onto gp120 by Dr. Peter Kwong). The residues important for the interaction with CD4 and the coreceptor are indicated in blue and green, respectively. B. Inhibition of virus variants with sCD4 and AMD3100 on CD4+ enriched lymphocytes. Inhibition curves are depicted for each of the viruses in limiting dilutions of either sCD4 (left panel) or the CXCR4 inhibitor AMD3100 (right panel).
Mentions: To assess the conformation and function of the mutant and revertant Env proteins on virus particles, we studied the sensitivity of the revertant viruses to inhibitors of viral entry in a PBMC-based neutralization assay (fig. 4). The location of the binding sites of the receptor, CD4, and the CXCR4 coreceptor for HIV-1LAI in relation to the positions of the mutations and reversions in our viruses is given in fig. 4A. Soluble CD4 (sCD4) was used to assess the interaction of these viruses with CD4. The wt virus was neutralized by sCD4 with an IC50 of ~16 μg/ml. The various viruses (except mut, which did not replicate in PBMCs), were neutralized efficiently at lower concentrations of sCD4 (IC50 of ~1–8 μg/ml) suggesting that the affinity for CD4 is increased in these variants. R1b was the most sensitive to neutralization by sCD4 (IC50 of ~1.0 μg/ml).

Bottom Line: In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12.Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent.Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Experimental Virology, Dept, Medical Microbiology, Center of Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands. r.w.sanders@amc.uva.nl

ABSTRACT

Background: The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for approximately 50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain.

Results: The 386 carbohydrate was not essential for folding of wt gp120. However, its removal improved folding of a gp120 variant lacking the 385-418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies.

Conclusion: The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.

Show MeSH
Related in: MedlinePlus