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：正链引发和整合。用莫洛尼鼠白血病 在逆转录病毒（M-MuLV）中，RNase H在正链引发中的作用 转录已被定义。一个类似的体外系统来研究艾滋病毒 将设置正链预充。突变分析将用于 确定HIV多嘌呤序列的那些特征， 重要的是定位RNase H的切割反应， 产生RNA引物。将在体内检测选定的M-MULV突变体 确认体外结果并进一步确定特异性 裂解反应的决定因素。DNA寡核苷酸， 与M-MuLV和HIV多嘌呤片段互补的多嘌呤片段将退火， 作为断裂底物测试的各病毒RNA和复合物 在体外对于相应的RNase H酶。如果特定的破损是 观察到，随后将测试寡核苷酸作为潜在的 在感染细胞中的逆转录抑制剂。这种做法 可能会导致开发出基于阿托西肽的药物， 用于治疗HIV感染者。 在初步实验中，M-MULV-1基因的编码M-MULV-1基因的区域 已经克隆了整合所需的蛋白质（IN蛋白）， 表达在“E.杆菌将纯化M-MuLV和HIV IN蛋白 和表征了特异性寡核苷酸将用于研究 蛋白质与已知参与的DNA序列的相互作用 一体化整合反应可以分为两个步骤： 前病毒DNA末端的切割和切割的DNA的附着 在交错断裂的位置与靶DNA结合。特殊DNA底物 将被设计成将反应的两个步骤进行生物化学分离 目的是确定反应的机理和 IN蛋白质

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.