700 Increased CD4 Affinity and Chemokine Receptor Binding Site Exposure in the gp120 of an In Vitro Microglia-Adapted HIV-1 Isolate J. Martin-Garcia*, D. van Ryk, F. Gonzalez-Scarano Univ of Pennsylvania Sch Med, Philadelphia
Background: Microglial cells, the main target of HIV-1 in the CNS, express low levels of CD4 in comparison to PBMCs. We have reported that the envelope glycoprotein from HIV-1Bori-15, a virus adapted in vitro to the growth in microglia, as compared to the envelope of the primary, blood-derived parental virus (HIV-1Bori), features: 1) higher ability to use low levels of CD4 for infection; 2) more prominent syncytium-inducing phenotype; and 3) greater exposure of the 17b epitope that correlates with higher neutralization sensitivity. In this study, our aim was to evaluate if the phenotypic changes were associated with an increased affinity in the binding to CD4 and the MAb whose epitopes co-localize with the chemokine receptor binding site.
Methods: We produced recombinant soluble proteins (sgp120) from both isolates, and used them in ELISA, FACS and surface plasmon resonance experiments to analyze binding to sCD4, CD4-pseudotyped MLV particles, HeLa cell clones expressing varying CD4 levels, and purified human 17b and 48d MAb.
Results: ELISA/FACS experiments did not demonstrate differences in the binding of sgp120s to sCD4, MLV-CD4, or HeLa-CD4 clones. Binding of 17b MAb was also identical to both sgp120s independently of the presence or absence of sCD4. However, 48d MAb binding to Bori-15 sgp120 was slightly greater than to Bori sgp120. We then performed surface plasmon resonance binding experiments and found that although Bori sgp120 (similar to a control BaL sgp120) bound to immobilized sCD4 with higher affinity that Bori-15 at 25°C, when the binding experiments were performed at 37°C, Bori-15 sgp120 bound to sCD4 with more than 50 times greater affinity than Bori (according to the affinity dissociation constant). Binding of Bori-15 sgp120 to 17b and 48d MAb in the absence of sCD4 was much greater than that of Bori or BaL sgp120s. Since the maximum binding to 17b and 48d MAb in the presence of saturating concentrations of sCD4 was comparable with the 3 proteins, we analyzed the percentage of maximum binding obtained in the absence of sCD4, which was higher in Bori-15 (26.4% and 14.4%) than in Bori (15.0% and 5.8%) or BaL (6.6% and 3.1%).
Conclusions: In vitro adaptation to the growth in purified microglial cultures was associated with the generation of a virus with an envelope glycoprotein featuring increased affinity for CD4 and a greater exposure of the conserved epitopes that co-localize with the chemokine receptor binding site on gp120.
