ASSAYING THE ACTIVITY OF HIV-1 INTEGRASE WITH DNA-COATED PLATES

Integration of reverse transcribed viral DNA of HIV into host chromoso mes is mediated by the viral enzyme, integrase. This enzymatic activit y can be monitored in vitro by integration of a small labeled DNA (don or) into a second unlabeled DNA (target). The methodology usually invo lves isotope labeling and gel electrophoresis. To simplify the measure ment, a method mimicking enzyme-linked immunosorbent assay (ELISA) pro cedures was developed. Fragments of DNA were adsorbed directly on 96-w ell plates and used as the target DNA. The donor was a synthetic 21-bp DNA duplex of HIV-1 U5 LTR; biotin was incorporated into the 5' end o f one strand whose two nucleotides at the 3' end were specifically rem oved during the integration. As a result of integration, the biotin-la beled donor DNA was joined with the target DNA and became immobilized on plates. These integration products were then measured by binding of avidin-alkaline phosphatase on plates. The method is simple and strai ghtforward and can easily be adapted for high throughput screening of integrase inhibitors.