Identification of the optimal DC-SIGN binding site on human immunodeficiency virus type 1 gp120.
Human immunodeficiency virus type 1 (HIV-1) envelope (gp120) binding to DC-SIGN, a C-type lectin that can facilitate HIV infection in cis and in trans, is largely dependent on high-mannose-content moieties. Here, we delineate the N-linked glycosylation (N-glycan) sites in gp120 that contribute to optimal DC-SIGN binding. 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 2G12. Consistent with this observation, HIV strain JRCSF gp120 prebound to 2G12 was 10-fold more sensitive to mannan competition than gp120 that was not prebound in a DC-SIGN cell surface binding assay. The analysis of multiple mutant forms of the 2G12 epitope revealed one triple glycosylation mutant form, termed 134mut (carrying N293Q, N382Q, and N388Q mutations), that exhibited a significant increase in sensitivity to both mannan competition and endoglycosidase H digestion compared to that of the 124mut form (carrying N293Q, N328Q, and N388Q mutations) 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 form of gp120 also exhibited decreased binding to DC-SIGN in the context of native envelope spikes on a virion, and virus bearing 134mut exhibited less efficient DC-SIGN-mediated infection in trans. Significantly, 124mut and 134mut differed by only one glycosylation site mutation in each construct, and both 124mut and 134mut 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.
Hong
Patrick W-P
PW
Department of Microbiology, Immunology, and Molecular Genetics, 3825 MSB, UCLA, 609 Charles E. Young Dr. East, Los Angeles, CA 90095, USA.
Nguyen
Sandra
S
Young
Sophia
S
Su
Stephen V
SV
Lee
Benhur
B
D001665:Binding Sites; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D000939:Epitopes; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
D015815:Cell Adhesion Molecules; D000939:Epitopes; D015699:HIV Envelope Protein gp120; D037181:Lectins, C-Type; D011134:Polysaccharides; D011956:Receptors, Cell Surface; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin