Integration complexes derived from HIV vectors for rapid assays in vitro

Of three enzymes encoded by HIV-reverse transcriptase, protease, and integr ase-only the first two have been exploited clinically as inhibitor targets. Efforts to develop inhibitors of purified integrase protein have yielded m any compounds, but none with clinical utility. A different source of integr ation activity for studies in vitro is provided by replication intermediate s isolated from HIV-infected cells. These preintegration complexes (PICs) c an direct integration of the endogenously synthesized viral cDNA into an ad ded target DNA in vitro. Despite their authentic activities, assays of PICs have not been widely used due to technical obstacles, particularly the req uirement for handling large amounts of infectious HIV. Here, we describe gr eatly improved methods for producing PICs using HIV-based vectors that are capable of establishing an integrated provirus but not a spreading infectio n. We also report the development of a PIC integration assay using DNA-coat ed microtiter plates, which speeds assays of PIC integration in vitro. We u sed this method to screen a library of chemicals related to known integrase inhibitors and found a new compound, quinalizarin sulfate, that displayed enhanced activity against PICs.