PLUS-STRAND PRIMING AND INTEGRATION BY M-MULV & HIV

这项建议涉及逆转录病毒复制的两个方面 循环：正链启动和整合。利用Moloney小鼠白血病 病毒(M-MuLV)，核糖核酸酶H在反向正链启动中的作用 转录已经被定义了。一种类似的研究艾滋病毒的体外系统 将设置正链引爆。突变分析将被用来 确定HIV多尿路序列的那些特征 在定位RNaseH以进行切割反应中很重要 产生RNA引物。选定的M-MULV突变体将在体内进行测试 以确认体外研究结果并进一步确定其特异性 切割反应的决定因素。DNA寡核苷酸是 对M-MuLV和HIV多尿路的补充将被退火化到 分别测试作为破坏底物的病毒RNA和复合体 在体外寻找相应的RNaseH酶。如果特定破损是 观察到，这些寡核苷酸随后将作为潜在的 被感染细胞中的逆转录抑制物。这样一种方法 可能会导致基于寡核苷酸的药物的开发，这种药物可能 用于治疗艾滋病毒感染者。 在初步实验中，编码M-MULV“I”基因的区域 一种整合所需的蛋白质(IN蛋白质)已被克隆并 在大肠杆菌中表达。M-MuLV和HIV IN蛋白都将得到纯化 并以此为特征。将使用特定的寡核苷酸来研究 蛋白质与已知参与的DNA序列的相互作用 整合。整合反应可分为两个步骤： 前病毒DNA末端的切割和切割后DNA的附着 到交错断裂地点的目标DNA。特殊DNA底物 将被设计成以生化方式分离反应的两个步骤 目的是为了确定反应的机理和作用 IN蛋白。

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.