Human Immunodeficiency Virus Envelope (gp120) Binding to DC-SIGN and Primary Dendritic Cells Is Carbohydrate Dependent but Does Not Involve 2G12 or Cyanovirin Binding Sites: Implications for Structural Analyses of gp120-DC-SIGN Binding

Abstract

The calcium-dependent lectin, DC-SIGN, binds to human immunodeficiency virus (HIV) (and simian immunodeficiency virus) gp120 and mediates the binding and transfer of HIV from monocyte-derived dendritic cells (MDDCs) to permissive T cells. However, it has been recently reported that DC-SIGN binding to HIV gp120 may be carbohydrate independent. Here, we formally demonstrate that gp120 binding to DC-SIGN and MDDCs is largely if not wholly carbohydrate dependent. Endo-␤-N-glucosaminidase H (EndoH) treatment of gp120-Fc under conditions that maintained wild-type CD4 binding-and the full complement of complex glycans-significantly decreased (>90%) binding to DC-SIGN expressing cell lines, as well as to MDDCs. Any residual binding of EndoH-treated gp120-Fc to DC-SIGN was completely competed off with mannan. Mutational analysis indicated that no single glycosylation site affected the ability of gp120-Fc to bind DC-SIGN. To further guide our efforts in mapping the DC-SIGN binding sites on gp120, we used two well-characterized HIV inhibitory agents (2G12 monoclonal antibody and cyanovirin) that bind to high-mannose sugars on gp120. We showed that 2G12 and DC-SIGN bound to nonoverlapping sites in gp120 because (i) 2G12 did not block soluble gp120 or virion binding to DC-SIGN, (ii) 2G12 bound to gp120-Fc that was prebound to cell surface DC-SIGN, and (iii) gp120-Fc mutants that lack glycosylation sites involved in 2G12's epitope were also fully capable of binding DC-SIGN. These data were substantiated by the inability of cyanovirin to block gp120-Fc binding to DC-SIGN. Cyanovirin has been shown to effectively compete for 2G12 binding to gp120. Indeed, high concentrations of cyanovirin dramatically enhanced gp120-Fc binding to cell surfaces in the presence or absence of DC-SIGN. We provide evidence that this enhancement may be due to cyanovirin's ability to bridge gp120 to mannosylated cell surface proteins. These results have implications for antiviral therapeutics and for ongoing efforts to finely map the glycan structures on gp120 responsible for DC-SIGN binding.

L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis C virus

“…However, binding was not inhibited by mAbs to other regions of the SIGN molecules or by anti-E2 mAbs, at least when used individually. Similar patterns of inhibition have been observed for HIV-1 gp120, whose binding to DC-SIGN has recently been shown to be mediated by high-mannose sugars (21). There are potential conformational differences between the purified glycoproteins (E2 or gp120) and the virion-associated oligomers to consider; however, the glycan-dependence of HIV and HCV binding to L-SIGN and DC-SIGN may cause the interactions to be less dependent on protein conformation.…”

“…However, the specificity of these interactions is underscored by the observation that several viruses have envelopes that are glycosylated yet show little or no avidity for DC-SIGN and L-SIGN (11,21). The findings in this report indicate that HCV has coopted host glycosylation pathways in ways that promote binding to L-SIGN and DC-SIGN.…”

“…This finding is consistent with lectin recognition of glycans distributed over the surface of E2, and the attendant difficulty of blocking such interactions with monospecific agents. Similarly, gp120 binding to DC-SIGN is not blocked either by anti-gp120 mAbs or by mutation of individual N-linked glycosylation sites on gp120 (21).…”

L-SIGN (CD209L) and DC-SIGN (CD209) mediate transinfection of liver cells by hepatitis C virus

“…HCV capture by SIGN molecules depends on the presence of the CRD, indicating that recognition of high mannose oligosaccharides in the viral envelope glycoproteins is critical for binding. The specificity of this interaction is underscored by observations that (i) other C type lectins, such as langerin, CD23, and CLEC-1͞2, do not bind HCV E2 (45,46); (ii) glycosylated envelope proteins of several viruses show little or no avidity for SIGN molecules (36,47); and (iii) anti-L-SIGN and anti-DC-SIGN mAbs as well as mannan inhibit soluble E2 and HCV capture.…”

HIV-1 Transmission by Dendritic Cell-specific ICAM-3-grabbing Nonintegrin (DC-SIGN) Is Regulated by Determinants in the Carbohydrate Recognition Domain That Are Absent in Liver/Lymph Node-SIGN (L-SIGN)

“…Binding of HIV-1 particles to DC-SIGN is dependent on the interaction of the CRD with the N-linked high mannose oligosaccharides that decorate the HIV-1 envelope (Env) glycoprotein (17,36,37). HIV-1 capture and transmission can be significantly inhibited by soluble mannan, and deglycosylation of Env can also impair binding to DC-SIGN (29,34,38,39). DC-SIGN-mediated HIV-1 transmission is cell type-dependent and requires cell-cell contact (35, 39 -41).…”