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J. Virol. doi:10.1128/JVI.01765-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Optimal DC-SIGN binding to HIV-1 gp120 involves specific N-glycans within the 2G12 epitope

Department of Microbiology, Immunology & Molecular Genetics, Department of Pathology & Laboratory Medicine, UCLA AIDS Institute, UCLA School of Medicine, Los Angeles, CA 90095

^* To whom correspondence should be addressed. Email: bleebhl{at}ucla.edu.

	Abstract

HIV-1 envelope (gp120) binding to DC-SIGN, a C-type lectin that can facilitate human immunodeficiency virus (HIV) infection in cis and in trans, is largely dependent on high mannose moieties. Here, we delineate the N-linked glycosylation (N-glycan) sites in gp120 that contribute optimally to DC-SIGN binding. The soluble DC-SIGN was able to block 2G12 binding to gp120, but not vice versa, suggesting that DC-SIGN binds to a more flexible combination of N-glycans than does 2G12. Consistent with this observation, JRCSF gp120 pre-bound to 2G12 was ten-fold more sensitive to mannan competition when tested in a DC-SIGN cell surface binding assay. Analysis of multiple 2G12 epitope mutants revealed one triple glycosylation mutant, termed 134mut (N293Q/N382Q/N388Q), that exhibited a significant increase in sensitivity to both mannan competition and EndoH digestion, when compared to 124mut (N293Q/N328Q/N388Q) and wild-type gp120 in a DC-SIGN binding assay. Importantly, no such differences were observed when binding to Galanthus nivalis was assessed. The 134mut also exhibited decreased binding to DC-SIGN in the context of native envelope spikes on a virion, and exhibited less efficient DC-SIGN-mediated infection in trans. Significantly, 124mut and 134mut differed by only one glycosylation site, and both viruses exhibited wild-type levels of infectivity when used in a direct infection assay. In summary, while DC-SIGN can bind to a flexible combination of N-glycans on gp120, its optimal binding site overlaps with specific N-glycans within the 2G12 epitope. Conformationally intact envelopes that are DC-SIGN binding deficient can be used to probe the in vivo biological functions of DC-SIGN.

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