CHARACTERIZATION OF NEUTRALIZATION EPITOPES IN THE V2 REGION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-I GP120 - ROLE OF GLYCOSYLATION IN THE CORRECT FOLDING OF THE V1 V2 DOMAIN/

A number of monoclonal antibodies (MAbs) with various levels of neutra lizing activity that recognize epitopes in the V1/V2 domain of LAI-rel ated gp120s have been described. These include rodent antibodies direc ted against linear and conformational epitopes and a chimpanzee MAb, C 108G, with extremely potent neutralizing activity directed against a g lycan-dependent epitope. A fusion glycoprotein expression system that expressed the isolated V1/V2 domain of gp120 in native form was used t o analyze the structural characteristics of these epitopes. A number o f MAbs (C108G, G3-4, 684-238, SC258, 11/68b, 38/66a, 38/66c, 38/62c, a nd CRA3) that did not bind,vith high affinity to peptides immunoprecip itated a fusion glycoprotein expressing the V1/V2 domain of HXB2 gp120 in the absence of other human immunodeficiency virus sequences, estab lishing that their epitopes were fully specified within this region. B iochemical analyses indicated that in the majority of V1/V2 fusion mol ecules only five of the six glycosylation signals in the V1/V2 domain were utilized, and the glycoforms were found to be differentially reco gnized by particular MAbs. Both C108G and MAbs directed against confor mational epitopes reacted with large fractions of the fully glycosylat ed molecules but with only small fractions of the incompletely glycosy lated molecules. Mutational analysis of the V1 and V2 glycosylation si gnals indicated that in most cases the unutilized site was located eit her at position 156 or at position 160, suggesting the occurrence of c ompetition for glycan addition at these neighboring positions. Mutatio n of glycosylation site 160 destroyed the C108G epitope but increased the fraction of the molecules that presented the conformational epitop es, while mutation of the highly conserved glycosylation site at posit ion 156 greatly diminished the expression of the conformational epitop es and increased expression of the C108G epitope. Similar heterogeneit y in glycosylation was also observed when the HXB2 V1/V2 fusion glycop rotein was expressed without most of the gp70 carrier protein, and thu s, this appeared to be an intrinsic property of the V1/V2 domain. Hete rogeneity in expression of conformational and glycan-dependent epitope s was also observed for the natural viral env precursor, gPrl60, but n ot for gp120. These results suggested that the closely spaced glycosyl ation sites 156 and 160 are often alternatively utilized and that the pattern of glycosylation at these positions affects the formation of t he conformational structures needed for both expression of native epit opes in this region and processing of gPr160 to mature env products.