This proposal is concerned with two aspects of the retroviral replication cycle: plus-strand priming and integration. Using Moloney murine leukemia virus (M-MuLV), the role of RNase H in plus-strand priming during reverse transcription has been defined. A similar in vitro system to study HIV plus-strand priming will be set up. A mutational analysis will be used to determine those features of the HIV polypurine tract sequence that are important in positioning the RNase H for the cleavage reaction that generates the RNA primer. Selected M-MULV mutants will be tested in vivo to confirm the in vitro findings and further define the specificity determinants for the cleavage reaction. DNA oligonucleotides that are complementary to the M-MuLV and HIV polypurine tracts will be annealed to the respective viral RNAs and the complexes tested as breakage substrates in vitro for the corresponding RNase H enzymes. If specific breakage is observed, the oligonucleotides will subsequently be tested as potential inhibitors of reverse transcription in infected cells. Such an approach might lead to the development of oligonucleotide-based drugs that could be used in the treatment of HIV-infected individuals. In preliminary experiments, the region of the M-MULV """"""""I gene that codes for a protein required for integration (IN protein) has been cloned and expressed in """"""""E. coli. Both the M-MuLV and HIV IN proteins will be purified and characterized. Specific oligonucleotides will be used to study the interaction of the proteins with the DNA sequences known to be involved in integration. The integration reaction can be separated into two steps: cleavage of the ends of the proviral DNA and attachment of the cleaved DNA to the target DNA at the site of a staggered break. Special DNA substrates will be designed to biochemically separate the two steps of the reaction with the aim of determining the mechanism of the reaction and the role of the IN protein.
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