Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens

Abstract

The envelope spike of HIV is one of the most highly N-glycosylated structures found in nature. However, despite extensive research revealing essential functional roles in infection and immune evasion, the chemical structures of the glycans on the native viral envelope glycoprotein gp120--as opposed to recombinantly generated gp120--have not been described. Here, we report on the identity of the N-linked glycans from primary isolates of HIV-1 (clades A, B, and C) and from the simian immunodeficiency virus. MS analysis reveals a remarkably simple and highly conserved virus-specific glycan profile almost entirely devoid of medial Golgi-mediated processing. In stark contrast to recombinant gp120, which shows extensive exposure to cellular glycosylation enzymes (>70% complex type glycans), the native envelope shows barely detectable processing beyond the biosynthetic intermediate Man5GlcNAc2 (<2% complex type glycans). This oligomannose (Man5-9GlcNAc2) profile is conserved across primary isolates and geographically divergent clades but is not reflected in the current generation of gp120 antigens used for vaccine trials. In the context of vaccine design, we also note that Manalpha1-->2Man-terminating glycans (Man6-9GlcNAc2) of the type recognized by the broadly neutralizing anti-HIV antibody 2G12 are 3-fold more abundant on the native envelope than on the recombinant monomer and are also found on isolates not neutralized by 2G12. The Manalpha1-->2Man residues of gp120 therefore provide a vaccine target that is physically larger and antigenically more conserved than the 2G12 epitope itself. This study revises and extends our understanding of the glycan shield of HIV with implications for AIDS vaccine design.

Fig. 1.

MALDI-TOF MS analyses of the N-linked glycans released from gp120_JRCSF. Spectra of released N-linked glycans ([M + Na]⁺ ions) from desialylated recombinant gp120 expressed in HEK 293T cells (A), native HIV envelope derived from pseudovirions from HEK 293T cells (B), recombinant gp120 expressed in GnT I-deficient HEK 293S cells (C), native HIV envelope derived from pseudovirions from GnT I-deficient HEK 293S cells (D), and desialylated envelope isolated from HEK 293T culture supernatant after removal of virions (E) are illustrated. Representative glycan structures are shown for masses that may contain multiple isobaric structures. The full assignment of A and E is presented in SI Appendix, Table S1. Symbols used for the structural formulae in this and subsequent figures are as follows: ◇, Gal; ■, GlcNAc; ○, Man; , Fuc; ⋆, NeuNAc (50). The linkage position is shown by the angle of the lines linking the sugar residues (vertical line, two-link; forward slash, three-link; horizontal line, four-link; back slash, six-link). Anomericity is indicated by full lines for β-bonds and by broken lines for α-bonds (50).

Fig. 2.

MALDI-TOF MS analysis of released N-linked glycans from native HIV envelope. 92RW020 (clade A), DU422 (clade C), and SIV_mac239 isolates derived from pseudovirions from HEK 293T cells (A) and JRCSF isolate derived from pseudovirions from PBMCs (B) are shown.

Fig. 3.

Kinetics of ER α-mannosidase I processing of gp120. Two glycoforms were used: an ER gp120 glycoform bearing predominantly the Man₉GlcNAc₂ glycan (expressed in the presence of kifunensine) and an IC/cis-Golgi glycoform exhibiting predominantly Man₅GlcNAc₂ glycans, with a residual Man_6–9GlcNAc₂ population (expressed in GnT I-deficient cells). The rates of mannose hydrolysis were determined by MALDI-TOF MS analysis of released N-linked glycans ([M + Na]⁺ ions): Abundances of Man₉GlcNAc₂ (◆), Man₈GlcNAc₂ (□), and Man₅GlcNAc₂ (○) were recorded. (A) Hydrolysis of the ER gp120 glycoform was monitored in the presence of 1 μg (solid line) or 5 μg (dashed line) of ER α-mannosidase I. (B) MALDI-TOF MS for the released N-linked glycans for initial (t = 0) and processed sample (t = 120 min) abundances of Man₉GlcNAc₂ (m/z = 1,905.5) and Man₈GlcNAc₂ (m/z = 1,743.5) is shown for this ER gp120 glycoform. (C) Hydrolysis of the IC/cis-Golgi gp120 was monitored in the presence of 1 μg of ER α-mannosidase I. (D) MALDI-TOF MS for the released N-linked glycans for initial (t = 0) and processed sample (t = 120 min) abundances of Man₉GlcNAc₂ (m/z = 1,905.6) and Man₈GlcNAc₂ (m/z = 1,743.6) is shown for this IC/cis-Golgi gp120 glycoform.

Fig. 4.

Mannose patches of recombinant monomeric (Monomer) and virion-associated trimeric (Trimer) gp120. The glycosylation of monomeric gp120 reveals an unprocessed intrinsic patch of Man_5–9GlcNAc₂ glycans (Fig. 1A), which appears to have resisted α-mannosidase activity, consistent with the results from the kinetic study (Fig. 3). Within this patch, in some isolates of HIV, the precise arrangement of Manα1→2Man-terminating oligomannose glycans supports binding of 2G12. The remaining glycans in the monomer are a cell-specific mixture of complex type glycans. The trimer contains the original mannose patch, as evidenced by the neutralization of functional virus by 2G12. The array of complex glycans is absent in the native trimer, however, and appears to be replaced by a further range of Man_5–9GlcNAc₂ glycans (Figs. 1C and 2 A and B).