Background:  The calcium-dependent lectin, DC-SIGN, binds to HIV (and simian immunodeficiency virus) gp120 and mediates the binding and transfer of HIV from monocyte-derived dendritic cells (MDDC) to permissive T cells. Moreover, gp120 binding to DC-SIGN and MDDC is largely dependent on high mannose moieties, and initial mutational analyses suggested that DC-SIGN binds to a flexible combination of the 12 N-linked glycosylation sites in gp120 known to give rise to high mannose structures. Here, we used a series of more than 40 glycosylation mutants and additional competition assays to further delineate the N-linked glycosylation sites in gp120 that contribute preferentially to virus binding to DC-SIGN.

Methods:  The ability of wild type and mutant gp120-Fc to bind cell surface DC-SIGN under increasing concentrations of mannan or EndoH was detected by a FACS-based assay. The extra-cellular domain of DC-SIGN (ECD), which contains the carbohydrate recognition and oligomerization domains was also tested for gp120 binding under similar conditions in an ELISA-based assay. Viruses expressing the full-length mutant envelopes were tested for their ability to bind to DC-SIGN expressing cells as well as to recombinant ECD.

Results:  JRCSF gp120-Fc pre-bound to 2G12 (~20 mg/mL) was 10-fold more sensitive to mannan competition when tested in a DC-SIGN cell surface binding assay, indicating that the N-linked glycosylation sites on gp120 involved in DC-SIGN binding overlap with 2G12 epitope. 2G12 is a monoclonal antibody whose epitope consists of 5 N-linked glycosylation sites (N293, N328, N382, N388, N393) that give rise to high mannose structures. Analysis of multiple mutants revealed 1 triple glycosylation mutant (N293Q/N382Q/N388Q) within 2G12 epitope that exhibited a significant increased sensitivity to both mannan competition and EndoH digestion in a DC-SIGN binding assay. gp120 mutations identified above (which diminished DC-SIGN binding) were then engineered back into the full-length Env gp160 (gp120/gp41). Indeed, virus containing the triple mutation (N293Q/N382Q/N388Q) was defective in binding to both ECD and cell surface DC-SIGN. To confirm that DC-SIGN/gp120 interaction overlaps with 2G12 epitope, we showed that ECD was able to block 2G12 binding to gp120, but 2G12 was not able to block ECD binding to gp120.

Conclusions:  DC-SIGN can bind to broader range of N-linked glycosylation sites than 2G12 on HIV gp120; however, its preferential binding site overlaps with the 2G12 epitope.