The goals of this project are to examine the integration reaction, in which human immunodeficiency virus (HIV) DNA is incorporated into cellular DNA, and to understand the role of integration in the pathogenesis of AIDS. Initial experiments proposed are to characterize integration in blood cells of HIV-infected individuals. Polymerase chain reaction (PCR) will be performed to measure the levels of integrated and unintegrated HIV DNA, the compartmentalization of HIV DNA between cytoplasm and nuclease, and the forms of unintegrated HIV DNA. The investigator anticipates that these data will localize the probable site in the integration pathway of any observed inefficiency of integration. The influence of cell type on integration will also be examined. The remaining sections of this application describes a combined genetic and biochemical approach that focuses on HIV integrase (IN). Infections with virions containing mutations at defined positions in the 3' region of the HIV pol (specifies viral reverse transcriptase) gene will be examined for specific defects in integration. Quantitative analysis will indicate whether integration is required for productive HIV infection. To begin to define domains of IN, mutated IN proteins will be compared with wild-type (wt) proteins for activity in assay systems that explore each step in the integration pathway, including DNA binding, endonuclease, covalent linkage and DNA joining. These assays will also be used to compare purified integrases from viruses with different pathogenic potential. The unique aspect of this plan involves expressing and purifying IN from visna virus, a lentivirus that is capable of productive infection without integration. Analysis of visna virus IN may provide insights into HIV IN which may be relatively inefficient at catalyzing integration.
