A novel assay for the DNA strand-transfer reaction of HIV-1 integrase

An essential step in the replication of retroviruses is the integration of a DNA copy of the viral genome into the genome of the host cell. Integration involves a series of well defined DNA cleavage and strand-transfer steps that are catalyzed by the virally encoded enzyme integrase (1-4). The sequence-specific endonucleolytic activity of integrase removes two nucleotides from the 3' end of each LTR sequence at either end of the double-stranded DNA provirus. In the subsequent strand-transfer reaction, integrase non-specifically nicks the host cell DNA, creating a staggered break whose 5' ends are coordinately ligated to the processed 3' ends of each viral LTR. Although repair of the resultant integrated product may require host-encoded cellular enzymes, integrase is the sole protein responsible for both the specific and non-specific endonucleolytic steps as well as DNA strand transfer. The enzyme's absolute requirement for propagation of HIV-1 in cell culture defines integrase as an attractive target for antiviral chemotherapeutic intervention (5). We designed the microtiter plate assay with the specific aim of developing a high volume, non-radioisotopic assay for HIV-1 integrase which uses unique oligonucleotides to represent the LTR donor and target DNA substrates.