title: Characterization of neutralization epitopes in the V2 region of human immunodeficiency virus type 1 gp120: role of glycosylation in the correct folding of the V1/V2 domain.
abstract: A number of monoclonal antibodies (MAbs) with various levels of neutralizing activity that recognize epitopes in the V1/V2 domain of LAI-related gp120s have been described. These include rodent antibodies directed against linear and conformational epitopes and a chimpanzee MAb, C108G, with extremely potent neutralizing activity directed against a glycan-dependent epitope. A fusion glycoprotein expression system that expressed the isolated V1/V2 domain of gp120 in native form was used to analyze the structural characteristics of these epitopes. A number of MAbs (C108G, G3-4, 684-238, SC258, 11/68b, 38/66a, 38/66c, 38/62c, and CRA3) that did not bind with high affinity to peptides immunoprecipitated a fusion glycoprotein expressing the V1/V2 domain of HXB2 gp120 in the absence of other human immunodeficiency virus sequences, establishing that their epitopes were fully specified within this region. Biochemical analyses indicated that in the majority of V1/V2 fusion molecules only five of the six glycosylation signals in the V1/V2 domain were utilized, and the glycoforms were found to be differentially recognized by particular MAbs. Both C108G and MAbs directed against conformational epitopes reacted with large fractions of the fully glycosylated molecules but with only small fractions of the incompletely glycosylated molecules. Mutational analysis of the V1 and V2 glycosylation signals indicated that in most cases the unutilized site was located either at position 156 or at position 160, suggesting the occurrence of competition for glycan addition at these neighboring positions. Mutation of glycosylation site 160 destroyed the C108G epitope but increased the fraction of the molecules that presented the conformational epitopes, while mutation of the highly conserved glycosylation site at position 156 greatly diminished the expression of the conformational epitopes and increased expression of the C108G epitope. Similar heterogeneity in glycosylation was also observed when the HXB2 V1/V2 fusion glycoprotein was expressed without most of the gp70 carrier protein, and thus, this appeared to be an intrinsic property of the V1/V2 domain. Heterogeneity in expression of conformational and glycan-dependent epitopes was also observed for the natural viral env precursor, gPr160, but not for gp120. These results suggested that the closely spaced glycosylation sites 156 and 160 are often alternatively utilized and that the pattern of glycosylation at these positions affects the formation of the conformational structures needed for both expression of native epitopes in this region and processing of gPr160 to mature env products.
authors.affiliation: Public Health Research Institute, New York, New York 10016.
authors.forename: Z
authors.initials: Z
authors.lastname: Wu
authors.forename: S C
authors.initials: SC
authors.lastname: Kayman
authors.forename: W
authors.initials: W
authors.lastname: Honnen
authors.forename: K
authors.initials: K
authors.lastname: Revesz
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: S
authors.initials: S
authors.lastname: Vijh-Warrier
authors.forename: S A
authors.initials: SA
authors.lastname: Tilley
authors.forename: J
authors.initials: J
authors.lastname: McKeating
authors.forename: C
authors.initials: C
authors.lastname: Shotton
authors.forename: A
authors.initials: A
authors.lastname: Pinter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D015686:Gene Products, env / Q000378:metabolism; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000276:immunology / Q000378:metabolism*; D019302:HIV Envelope Protein gp160; D015497:HIV-1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats
chemical_list: D000939:Epitopes; D015686:Gene Products, env; D015699:HIV Envelope Protein gp120; D019302:HIV Envelope Protein gp160; D010446:Peptide Fragments; D011134:Polysaccharides; D011498:Protein Precursors