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Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding

View Article: PubMed Central - PubMed

ABSTRACT

The surface envelope glycoprotein (SU) of Human immunodeficiency virus type 1 (HIV-1), gp120SU plays an essential role in virus binding to target CD4+ T-cells and is a major vaccine target. Gp120 has remarkably high levels of N-linked glycosylation and there is considerable evidence that this “glycan shield” can help protect the virus from antibody-mediated neutralization. In recent years, however, it has become clear that gp120 glycosylation can also be included in the targets of recognition by some of the most potent broadly neutralizing antibodies. Knowing the site-specific glycosylation of gp120 can facilitate the rational design of glycopeptide antigens for HIV vaccine development. While most prior studies have focused on glycan analysis of recombinant forms of gp120, here we report the first systematic glycosylation site analysis of gp120 derived from virions produced by infected T lymphoid cells and show that a single site is exclusively substituted with complex glycans. These results should help guide the design of vaccine immunogens.

No MeSH data available.


Complete gp160 amino acid alignment of HIV-1 BaL/SupT1-R5.BAL.AY713409 is the published reference sequence (GenBank accession number AY713409). Dashed lines show sequence identity to the reference sequence and amino acid polymorphisms are indicated by a single letter amino acid abbreviation. Key landmarks in gp160 are denoted above each region. Potential N-linked glycosylation sites are shaded. In two sequences (H11 and E1), a nucleotide frame shift lead to a premature stop codon (*). In a third sequence (C12) a nucleotide change in W754 also led to a premature stop codon (*).
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f6: Complete gp160 amino acid alignment of HIV-1 BaL/SupT1-R5.BAL.AY713409 is the published reference sequence (GenBank accession number AY713409). Dashed lines show sequence identity to the reference sequence and amino acid polymorphisms are indicated by a single letter amino acid abbreviation. Key landmarks in gp160 are denoted above each region. Potential N-linked glycosylation sites are shaded. In two sequences (H11 and E1), a nucleotide frame shift lead to a premature stop codon (*). In a third sequence (C12) a nucleotide change in W754 also led to a premature stop codon (*).

Mentions: Single genome amplification (SGA) was utilized to generate unique, independent sequences from cultured HIV-1 BaL/SUPT1-CCR5 cells, as previously described34. The entire 3′ half of the viral genome (including the entire vif, vpr, vpu, tat, rev env and nef genes) was amplified from DNA (Qiagen DNA Blood kits). PCR was performed using a limiting dilution approach where only one amplifiable molecule was present in each reaction. PCR was performed with 1 × PCR buffer, 2 mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate, 0.2 μM of each primer, and 0.025 U/μL Platinum Taq polymerase (ThermoFisher) in a 20 μL reaction. First round PCR was performed with sense primer HIVBK3F1 5′-ACAGCAGTACAAATGGCAGTATT-3′ and antisense primer HIVR3B3.R1 under the following conditions: 1 cycle of 94 °C for 2 min, 35 cycles at 94 °C for 15 sec, 55 °C for 30 sec, and 72 °C for 4 min, followed by a final extension of 72 °C for 10 min. Next, 1 μL from the first-round PCR product was added to a second-round PCR reaction that included the sense primer HIVBK3F2 5′-TGGAAAGGTGAAGGGGCAGTAGTAATAC-3′ and antisense primer HIVR3B6.R2 5′-TGAAGCACTCAAGGCAAGCTTTATTGAGGC-3′ performed under the same conditions used for first-round PCR, but with a total of 45 cycles. Correct sized amplicons were identified by agarose gel electrophoresis and directly sequenced with second round PCR primers and HIV specific primers using BigDye Terminator technology. Sequences were aligned using ClustalW and hand edited using MacClade 4.08. The entire gp160 amino acid alignment is shown in Fig. 6.


Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding
Complete gp160 amino acid alignment of HIV-1 BaL/SupT1-R5.BAL.AY713409 is the published reference sequence (GenBank accession number AY713409). Dashed lines show sequence identity to the reference sequence and amino acid polymorphisms are indicated by a single letter amino acid abbreviation. Key landmarks in gp160 are denoted above each region. Potential N-linked glycosylation sites are shaded. In two sequences (H11 and E1), a nucleotide frame shift lead to a premature stop codon (*). In a third sequence (C12) a nucleotide change in W754 also led to a premature stop codon (*).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015092&req=5

f6: Complete gp160 amino acid alignment of HIV-1 BaL/SupT1-R5.BAL.AY713409 is the published reference sequence (GenBank accession number AY713409). Dashed lines show sequence identity to the reference sequence and amino acid polymorphisms are indicated by a single letter amino acid abbreviation. Key landmarks in gp160 are denoted above each region. Potential N-linked glycosylation sites are shaded. In two sequences (H11 and E1), a nucleotide frame shift lead to a premature stop codon (*). In a third sequence (C12) a nucleotide change in W754 also led to a premature stop codon (*).
Mentions: Single genome amplification (SGA) was utilized to generate unique, independent sequences from cultured HIV-1 BaL/SUPT1-CCR5 cells, as previously described34. The entire 3′ half of the viral genome (including the entire vif, vpr, vpu, tat, rev env and nef genes) was amplified from DNA (Qiagen DNA Blood kits). PCR was performed using a limiting dilution approach where only one amplifiable molecule was present in each reaction. PCR was performed with 1 × PCR buffer, 2 mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate, 0.2 μM of each primer, and 0.025 U/μL Platinum Taq polymerase (ThermoFisher) in a 20 μL reaction. First round PCR was performed with sense primer HIVBK3F1 5′-ACAGCAGTACAAATGGCAGTATT-3′ and antisense primer HIVR3B3.R1 under the following conditions: 1 cycle of 94 °C for 2 min, 35 cycles at 94 °C for 15 sec, 55 °C for 30 sec, and 72 °C for 4 min, followed by a final extension of 72 °C for 10 min. Next, 1 μL from the first-round PCR product was added to a second-round PCR reaction that included the sense primer HIVBK3F2 5′-TGGAAAGGTGAAGGGGCAGTAGTAATAC-3′ and antisense primer HIVR3B6.R2 5′-TGAAGCACTCAAGGCAAGCTTTATTGAGGC-3′ performed under the same conditions used for first-round PCR, but with a total of 45 cycles. Correct sized amplicons were identified by agarose gel electrophoresis and directly sequenced with second round PCR primers and HIV specific primers using BigDye Terminator technology. Sequences were aligned using ClustalW and hand edited using MacClade 4.08. The entire gp160 amino acid alignment is shown in Fig. 6.

View Article: PubMed Central - PubMed

ABSTRACT

The surface envelope glycoprotein (SU) of Human immunodeficiency virus type 1 (HIV-1), gp120SU plays an essential role in virus binding to target CD4+ T-cells and is a major vaccine target. Gp120 has remarkably high levels of N-linked glycosylation and there is considerable evidence that this “glycan shield” can help protect the virus from antibody-mediated neutralization. In recent years, however, it has become clear that gp120 glycosylation can also be included in the targets of recognition by some of the most potent broadly neutralizing antibodies. Knowing the site-specific glycosylation of gp120 can facilitate the rational design of glycopeptide antigens for HIV vaccine development. While most prior studies have focused on glycan analysis of recombinant forms of gp120, here we report the first systematic glycosylation site analysis of gp120 derived from virions produced by infected T lymphoid cells and show that a single site is exclusively substituted with complex glycans. These results should help guide the design of vaccine immunogens.

No MeSH data available.