Gp120 on HIV-1 Virions Lacks O-Linked Carbohydrate

Abstract

As HIV-1-encoded envelope protein traverses the secretory pathway, it may be modified with N- and O-linked carbohydrate. When the gp120s of HIV-1 NL4-3, HIV-1 YU2, HIV-1 Bal, HIV-1 JRFL, and HIV-1 JRCSF were expressed as secreted proteins, the threonine at consensus position 499 was found to be O-glycosylated. For SIVmac239, the corresponding threonine was also glycosylated when gp120 was recombinantly expressed. Similarly-positioned, highly-conserved threonines in the influenza A virus H1N1 HA1 and H5N1 HA1 envelope proteins were also found to carry O-glycans when expressed as secreted proteins. In all cases, the threonines were modified predominantly with disialylated core 1 glycans, together with related core 1 and core 2 structures. Secreted HIV-1 gp140 was modified to a lesser extent with mainly monosialylated core 1 O-glycans, suggesting that the ectodomain of the gp41 transmembrane component may limit the accessibility of Thr499 to glycosyltransferases. In striking contrast to these findings, gp120 on purified virions of HIV-1 Bal and SIV CP-MAC lacked any detectable O-glycosylation of the C-terminal threonine. Our results indicate the absence of O-linked carbohydrates on Thr499 as it exists on the surface of virions and suggest caution in the interpretation of analyses of post-translational modifications that utilize recombinant forms of envelope protein.

Fig 1. Conservation of Thr for HIV-1 and HIV-2/SIVsm/SIVmac.

(A) Sequences were obtained from the HIV-1/SIVcpz alignment from the Los Alamos HIV database [5]. Sequences of Env from whole genomes of HIV-1 group M and HIV-1 group O that had a defined amino acid at HXB2 position 499 were analyzed. Shown is the rounded percentage of each amino acid: Thr (Thr), serine (Ser), asparagine (Asn), alanine (Ala), aspartic acid (Asp), proline (Pro), glycine (Gly), isoleucine (Ile), leucine (Leu), and tyrosine (Tyr). For the 3017 sequences, 2883 had Thr, 81 had Ser, 33 had Asn, 6 had Ala, 5 had Asp, 5 had Pro, and one sequence had Gly, Ile, Leu or Tyr. (B) Alignment of the C-terminus of gp120 and the N-terminus of gp41 for select viral species from the Lentivirus genus of the Retroviridae. Highly conserved Thr is denoted in red. The last R residue in black is taken as the C-terminus of gp120 by conventional usage. Light blue lettered amino acids are the N-terminus of gp41.

Fig 2. O-linked carbohydrate analyses of recombinant gp120 and gp120 purified from virions.

(A) Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectra of permethylated O-glycans isolated from HIV-1 NL4-3 gp120. All molecular ions are [M+Na]⁺. The sugar symbols are those employed by the Consortium for Functional Glycomics for the representation of glycan structures. Structural assignments are based on monosaccharide composition (obtained by MALDI-TOF MS), fragmentation analyses (MALDI-TOF/TOF MS/MS), and knowledge of glycan biosynthetic pathways. Asterisk denotes signals from contaminating N-glycans. (B) Electrospray Nano-LC-MS/MS spectra showing the location and attachment of the major species of O-glycan attached to Thr at position 497 in the truncated secreted HIV-1 NL4-3 gp120 (Thr499 in HXB2). (C) Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectra of permethylated O-glycans isolated from the truncated secreted SIVmac239 gp120 (D) Electrospray Nano-LC-MS/MS spectra showing the location and attachment of the major species of O-glycan attached to Thr at position 510 in the truncated secreted SIVmac239 gp120.

Fig 3. O-linked carbohydrate of recombinant gp120s from other strains of HIV-1.

Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectra of permethylated O-glycans isolated from gp120 of (A) HIV-1 JRCSF (B) HIV-1 YU2. See legend to Fig 1 for additional information. Signals that are not annotated are from impurities.

Fig 4. Conservation of a C-terminal threonine in HA1 of influenza A.

(A) Alignment of the C-terminus of HA1 and the N-terminus of HA2 for subtypes of influenza A virus. The highly conserved Thr is shown in red. Black letters are amino acids at the C-terminus of HA1. Light blue letters are amino acids at the N-terminus of HA2.

Fig 5. O-glycosylation status of the similarly positioned conserved Thr of influenza A virus H1N1 Puerto RIco HA1 and of influenza A virus H5N1 Thai HA1.

(A) MALDI-TOF MS of permethylated O-glycans isolated from recombinant HA1 of Influenza A virus H1N1 Puerto Rico (B) MALDI-TOF MS of permethylated O-glycans isolated from recombinant HA1 of Influenza A virus H5N1 Thai (C) Electrospray Nano-LC-MS/MS spectra showing the location and attachment of the major species of O-glycan attached to Thr318 in HA1 of Influenza A virus H1N1 strain Puerto Rico; parent ion [M+2H]²⁺ m/z 895.48. (D) Electrospray Nano-LC-MS/MS spectra showing the location and attachment of the major species of O-glycan attached to Thr318 in HA1 of Influenza A virus H5N1 strain Thai; parent ion [M+2H]²⁺ m/z 895.48.