The glycan shield of HIV is predominantly oligomannose independently of production system or viral clade

Abstract

The N-linked oligomannose glycans of HIV gp120 are a target for both microbicide and vaccine design. The extent of cross-clade conservation of HIV oligomannose glycans is therefore a critical consideration for the development of HIV prophylaxes. We measured the oligomannose content of virion-associated gp120 from primary virus from PBMCs for a range of viral isolates and showed cross-clade elevation (62-79%) of these glycans relative to recombinant, monomeric gp120 (∼30%). We also confirmed that pseudoviral production systems can give rise to notably elevated gp120 oligomannose levels (∼98%), compared to gp120 derived from a single-plasmid viral system using the HIV(LAI) backbone (56%). This study highlights differences in glycosylation between virion-associated and recombinant gp120.

Figure 1. Comparison of recombinant, pseudoviral and viral gp120.

MALDI-TOF MS analyses of released desialylated N-linked glycans ([M+Na]⁺ ions) from: (A) recombinant monomeric gp120_JRCSF expressed in HEK 293T cells; (B, C and D) respectively gp160_JRCSF, gp120_JRCSF and soluble, non-virion associated envelope gp120_JRCSF isolated from pseudoviral particle preparations generated by transfection of HEK 293T cells with the pSVIII-JRCSF and pSG3Δenv plasmids at a ratio of 1∶10; (E) gp120_JRCSF isolated from replication competent viral particles generated by transfection of HEK 293T cells with pLAI-JRCSF env molecular clone; (F, G and H) respectively gp120_92RW009, gp120_JRCSF and gp120_93IN905 isolated from virus obtained by infection of human PBMCs. Symbols used for the structural formulae in this and subsequent figures: ⋄  =  Gal, ▪  =  GlcNAc, ○  =  Man,  =  Fuc . The linkage position is shown by the angle of the lines linking the sugar residues (vertical line  = 2-link, forward slash  = 3-link, horizontal line  = 4-link, back slash  = 6-link). Anomericity is indicated by full lines for β-bonds and broken lines for α-bonds . The oligomannose series are highlighted.

Figure 2. Multiple divergences of gp120 glycosylation from host cell glycosylation.

Following removal of terminal α-linked glucose residues in the ER, folded glycoproteins contain exclusively oligomannose glycans. During transit through the ER, intermediate compartment (IC) and cis-Golgi apparatus, Manα1→2Man termini are removed by ER Mannosidase I and Golgi Mannosidases A–C to yield Man₅GlcNAc₂. However, the oligomannose cluster intrinsic to monomeric gp120 , limits glycan processing on both monomeric and oligomeric gp120 , . The steric consequences of trimerisation further limit Manα1→2Man trimming leading to an additional ‘trimer-associated’ population of Man_5–9GlcNAc₂. The exposed Man₅GlcNAc₂ glycans on gp120 that passage through the full extent of the Golgi apparatus and trans Golgi network (TGN) to the plasma membrane (PM) are processed by GnT I and subsequent enzymes to form complex-type glycans. However, envelope glycoprotein that does not follow this route to the PM is characterized by an elevated abundance of Man₅GlcNAc₂ (and closely resembles gp120 expressed in GnT I-deficient cells [11], [30]), and reduced furin cleavage. Thus the intrinsic mannose patch, which includes the 2G12 epitope, persists from the earliest stages of glycan processing whilst other elements of the glycan shield exhibit variably processed glycans depending on oligomerization state and, at least in the case of pseudoviral gp160/gp120, cellular trafficking.