Integration of a DNA copy of the viral genome into a chromosome of the host cell is a necessary step in the replication cycle of HIV and other retroviruses. The objectives of this project are to understand the detailed molecular mechanism of this step in the retroviral replication cycle. They key enzyme required for integration s the virally encoded integrase protein. Our research focusses on the biochemical activities of the HIV-1 integrase protein. Integrase is a site-specific nuclease that cleaves two nucleotides from each 3' end of the viral DNA prior to integration. Integrase also carries out the subsequent DNA strand transfer reaction that covalently inserts the viral DNA into the host genome. A major emphasis of the project is now to obtain detailed structural information on integrase. This has previously not been possible because the protein forms large heterogeneous aggregates, even at low concentration. We have made site-directed mutations in the catalytic core domain of HIV-1 integrase and identified several mutants with physical properties that make them better candidates for physical studies. One of these mutants is considerable more soluble than the wild type protein and retains catalytic activity. Structural studies of this protein are in progress. Viral DNA made in vivo by reverse transcription exists as a large nucleoprotein complex. Although integrase protein alone can carry out the enzymatic steps that insert viral DNA into host DNA in vitro, the integrity of this complex is likely to be important for the integration process in vivo. We are studying such complexes, isolated from cells infected with Moloney murine leukemia virus, to elucidate features of the integration process that have not been reproduced in simple reaction systems containing only integrase and synthetic DNA substrate. We find that the DNa within these complexes is protected against self-destructive integration into itself. This protection, which probably plays the physiological role of preventing autointegration of the viral DNA before the integration complex encounters host DNA, is conferred by a factor of cellular origin.
NIH ExPorter
NIH ExPorter