The goal of the proposed project is to better understand retroviral integration and to use that information to develop therapeutic integration inhibitors. We plan to achieve that goal with three specific avenues of investigation. The first and largest approach will be the development and use of an in vivo integration assay to i) validate the physiologic relevance of the recently acquired in vitro data from IN mutants, ii) identify IN mutants that are enzymatically active yet integration deficient (a class of mutants that cannot be identified by the current array of in vitro assays and which may provide new avenues for therapeutic intervention), and iii) determine which of the current in vitro assays of IN function -- 3' processing, strand transfer, disintegration, and target site selection - - correlates best with in vivo provirus formation. The latter issue is very important for assay development by our partners in the pharmaceutical industry; the in vitro assay they use to screen for integrate inhibitors must correlate well with in vivo provirus formation. Furthermore, the in vivo assay can be used to test candidate drugs found during mass screening in vitro. The second approach is the establishment of an in vitro integration assay that scores for the concerted integration of both ends of the HIV-1 DNA into a single site on the target DNA. Provirus formation requires that both ends of a single viral genome integrate in a concerted fashion at a single location on the host chromosome, but purified HIV-1 IN has never been shown to produce a concerted integration in vitro. An in vitro assay for HIV-1 IN specifically designed to score for concerted integration in vitro. An in vitro assay for HIV-1 In specifically designed to score for concerted integration should further our understanding of the integration process, possibly by defining important physical or chemical co-factors. The third approach is to use monoclonal antibodies against HIV-1 IN to help identify significant domains of IN. Defining domains by mutant analysis has been modestly illuminating, at best. Given the plethora of in vitro assays for the different IN activities, we can use well-mapped monoclonal antibodies to complement the mutagenesis data in an effort to identify functional domains of IN.
|Gaur, M; Leavitt, A D (1998) Mutations in the human immunodeficiency virus type 1 integrase D,D(35)E motif do not eliminate provirus formation. J Virol 72:4678-85|
|Leavitt, A D; Robles, G; Alesandro, N et al. (1996) Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection. J Virol 70:721-8|