Integration of a DNA copy of a retroviral genome into a chromosome of a infected cell is an essential step for normal viral replication. Our objectives are to analyze the detailed molecular mechanism of this DNA integration reaction and to develop simple cell-free assay systems that may be used to screen for drugs that inhibit this step of the viral replication cycle. Retroviral DNA integration involves two central steps, cleavage of two nucleotides from the 3' ends of the viral DNA and subsequent joining of these processed 3' ends to the 5' ends of a staggered cut made in the target DNA. We have determined with both the Moloney murine leukemia virus (MoMLV) and HIV systems that a single viral protein accomplishes these reactions. This protein is the IN protein which is encoded at the 3' end of the viral pol gene. Our current research therefor focusses on the biochemical activities of the MoMLV and HIV IN proteins. The MoMLV IN protein has been expressed in insect cells and partially purified. MoMLV IN protein has a site-specific nuclease activity that cleaves two nucleotides from the sequences present at the ends of MoMLV DNA. This reaction generates the recessed 3' ends that the precursors for integration. MoMLV IN protein also accomplishes the subsequent step of integrating these ends into a target DNA. We have expressed the HIV IN protein in insect cells and shown that it has the same biochemical activities as the MoMLV IN protein. To provide a more abundant source of HIV IN protein for physical studies we have also expressed this protein in E. coli and purified it in active form. We can now efficiently carry out the two central steps of retroviral DNA integration with cloned MoMLV or HIV IN proteins and oligonucleotide DNA substrates. This assay is being developed to provide a simple and economical means to screen for drugs that inhibit HIV DNA integration.