View All Abstracts for Session 24
119 Selective Mutations of the gp120 Glycosylations Sites on an Acute HIV-1 Infection Primary Isolate env Sequence According to their Clustering on the 3D Model
F. Bedin, Y. Ataman-Önal, F. Reynard, P.-Y. Durand, M. Girard, and B. Verrier*
CNRS-BioMerieux, CERVI, Lyon, France
Background: Carbohydrates comprise about 50% of the mass of the HIV-1 gp120. They contribute, among others, to divert immune response acting like a shield. Many studies have already been done concerning the biological relevance of the different glycosylation sites, but only a few have taken into account available 3D data with a systematic approach.
Methods: We compared 13 clade B primary env sequences (gp120) from patients with acute HIV-1 infection isolated and characterized in our laboratory (8 homosexual and 5 heterosexual transmissions). Potential glycosylation sites conserved on all sequences were identified. We hypothesized that these sites were also present in the transmitted isolate and therefore had a biological significance. These conserved sites were placed on the gp120-crystallized 3D model. Based on the 3D vicinity of these potential glycosylation sites, hotspots defining 4 “glycosylation clusters” were highlighted. According to this preliminary analysis we constructed by site-directed mutagenesis 18 partially deglycosylated env mutants all cloned into a eucaryotic expression vector. All constructs were tested both for their antigenicity (ELISA) and functionality (cleavage rate and syncytia formation assays on CD4+/CXCR4+ or CD4+/CCR5+ GHOST cells) .
Results: We observed a direct correlation among the number of mutations within a specific glycosylation cluster, the cleavage rate, the syncytia formation, and the recognition of the whole protein both by a pool of 3 HIV-1 sera (2 clade B and 1 clade C) and by a monoclonal antibody directed against the CD4-induced epitopes (CG10). Moreover, the soluble CD4 fixation fluctuated in the same way. Two of the most potent HIV-1 neutralizing antibodies identified to date (IgG1b12 and 2G12) did not react either with the wild-type or the mutants proteins. The same results were noticed on mutants constituted by 3 or 4 mutations picked at random from the 4 different clusters. Our results also indicated that some pool of mutations could completely affect the general structure of the gp120. Thus selective mutations of the gp120 glycosylation sites according to their clustering on the 3D model could modify its antigenicity.
Conclusions: These results deduced from this new approach could be helpful in the choice of partially deglycosylated Env proteins that truly mimic the native protein and may induce broad range neutralizing antibodies by unmasking conformational epitopes not yet characterized.
Contact Author about this Abstract
