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. 2018 Mar 6;115(10):2443-2448.
doi: 10.1073/pnas.1722627115. Epub 2018 Feb 20.

Signal Peptide of HIV Envelope Protein Impacts Glycosylation and Antigenicity of gp120

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Free PMC article

Signal Peptide of HIV Envelope Protein Impacts Glycosylation and Antigenicity of gp120

Jason Yolitz et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The HIV-1 envelope protein (Env) of early-replicating viruses encodes several distinct transmission signatures. One such signature involves a reduced number of potential N-linked glycosylation sites (PNGs). This transmission signature underscores the importance of posttranslational modifications in the fitness of early-replicating isolates. An additional signature in Env involves the overrepresentation of basic amino acid residues at a specific position in the Env signal peptide (SP). In this report, we investigated the potential impact of this SP signature on gp120 glycosylation and antigenicity. Two recombinant gp120s were constructed, one derived from an isolate that lacks this signature and a second from an early-replicating isolate that includes this signature. Chimeric gp120s were also constructed in which the two SPs were swapped between the isolates. All four gp120s were probed with glycan-, structure- and receptor- specific probes in a surface plasmon resonance binding assay. We found that the SP of Env influences qualitative aspects of Env glycosylation that in turn affect the antigenicity of Env in a major way. The SP impacts the affinity of Env for DC-SIGN, a lectin receptor expressed on dendritic cells that is believed to play a role in mucosal transmission. Additionally, affinity for the monoclonal antibodies 17b and A32, which recognize a CD4-induced, open conformation of Env is also altered. These results demonstrate that natural variation in the SP of HIV Env can significantly impact the antigenicity of mature gp120. Thus, the SP is likely subject to antibody-mediated immune pressure.

Keywords: HIV; antigenicity; gp120; signal peptide; vaccine.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
HIV Env SP role in early HIV Env processing and glycosylation. (A) N-linked glycans are added in the ER to the asparagine in an N-linked glycosylation sequon of nascent peptides as Glc3Man9GlcNAc2. The three Glcs are sequentially removed followed by removal of one Man residue. This glycosidase activity is intimately associated with glycoprotein folding assisted by ER resident chaperones and determines whether a protein traffics to the Golgi or is degraded by the proteasome. Once in the Golgi, the glycans are further modified with the addition of GlcNAc, Gal, Fuc, sialic acids, or other complex saccharide linkages. Gal, galactose; Glc, glucose; GlcNAc, N-acetyl glucosamine; Fuc, fucose; Man, mannose. (B) Sequence logo of natural variation found in 2,025 Clade B HIV Env SP sequences from the Los Alamos National Laboratory HIV Database using Weblogo 3. The height of each symbol indicates relative frequency of each amino acid at that position. Amino acid symbols are color coded for hydrophobicity: blue, hydrophilic; green, neutral; black, hydrophobic. (C) SP amino acid sequence alignment of the SP of the early-replicating isolate bearing the SP transmission signature and of the isolate that does not bear the SP transmission signature. Amino acid residues are color coded for side chain chemistry: orange, nonpolar; blue, basic; green, polar; yellow, aromatic; red, acidic.
Fig. 2.
Fig. 2.
HIV Env SP impacts recombinant gp120 molecular mass. Size exclusion chromatography (SEC) chromatograms of the recombinant (A) early-replicating gp120 with its wild-type SP (blue), the chronic SP (green), (B) the chronic gp120 with its wild-type SP (green), and the early-replicating SP (blue) produced in CHO-S cells and purified via Galanthus nivalis lectin column. (C) Tabulated molecular mass and molecular mass changes for all four proteins. w, with.
Fig. 3.
Fig. 3.
HIV Env SP influences gp120 glycosylation profile. Four recombinant gp120s were evaluated for glycan composition in a surface plasmon resonance (SPR) assay probing with (A) N. pseudonarcissus lectin specific for α-linked mannose residues and (B) R. communis I lectin specific for oligosaccharides ending in galactose. (C) The four gp120 were also probed with the glycan-dependent mAb 2G12. (Left) Sensograms of the early-replicating gp120 with wild-type SP (blue) and with the chronic SP (green), and (Right) sensograms of the chronic gp120 with its wild-type SP (green) and with the early-replicating SP (blue). Peak response units (RU) are presented and circled above each curve. w, with.
Fig. 4.
Fig. 4.
HIV Env SP influences DC-SIGN reactivity to gp120. (A) The early-replicating gp120 with its wild-type SP (blue) and with the chronic SP (green), and (B) the chronic gp120 with its wild-type SP (green) and the chronic gp120 with the early-replicating SP (blue) were evaluated for reactivity to soluble tetrameric DC-SIGN, C-type lectin receptor expressed on dendritic cells, in an SPR assay. Peak response units (RU) are presented and circled above each curve. w, with.
Fig. 5.
Fig. 5.
HIV Env signal peptide influences gp120 structure and antigenicity. (A) Subtype A gp120 92UG037 (blue) and the PNG mutant 92UG037 N144Q (green) were probed for reactivity with the CD4i mAbs 17b (Left) and A32 (Right) in an SPR assay to determine the impact of glycosylation on gp120 antigenicity. (B) Early-replicating gp120 with wild-type SP (blue) and early-replicating gp120 with the chronic SP (green) were probed with mAbs 17b (Left) and A32 (Right) in an SPR assay. (C) Chronic gp120 with chronic SP (green) and chronic gp120 with the early-replicating SP (blue) were also probed with the mAbs 17b (Left) and A32 (Right). Peak response units (RU) are presented and circled above each curve. w, with.

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