IN VITRO REPLICATION OF AN HIV DNA INTERMEDIATE

这一建议侧重于合成和可能调节的信号 将HIV的(+)链DNA中间体18个碱基终止于 用于(-)链DNA合成的tRNA/lys3引物。我提议一项 TRNA/lys3第58位甲基化的腺嘌呤可能会破坏 HIV的聚合酶，从而防止额外序列的复制 这可能会损害艾滋病毒的增长。建议进行实验以确定 修饰碱基在产生HIV(+)链DNA中间体中的作用 使用含有甲基化A58的兔肝脏tRNA/lys3与 T7RNA聚合酶产生的未修饰的tRNA/lys3。因为次要 TRNA/lys3中的结构也可能导致合成的RNA引物终止 具有相同3‘序列但没有天然二级结构的将是 测试以确定端接是否基于结构。最后， 我们实验室的初步观察表明，p51亚基 最初被认为是不活跃的逆转录酶，确实产生了 RNA引发的DNA模板上的DNA。因此，突变的p51将混合在一起。 以评估其在产生(+)链DNA中的作用 中级的。这些研究应有助于确定关键控制点 在艾滋病毒生命周期中，可能是治疗的潜在靶点 战略。

This proposal focuses on the synthesis and the signals that may regulate termination of the (+) strand DNA intermediate of HIV, 18 bases into the tRNA/lys3 primer for (-) strand DNA synthesis. I propose that a methylated adenine at position 58 of tRNA/lys3 may destabilize the polymerase of HIV thereby preventing replication of additional sequence that could impair HIV growth. Experiments are proposed to determine the role of modified bases in generating the HIV (+) strand DNA intermediate, using tRNA/lys3 from rabbit liver with a methylated A58 compared to unmodified tRNA/lys3 produced by T7 RNA polymerase. Because secondary structure in tRNA/lys3 may also cause termination, a synthetic RNA primer with the same 3' sequence but without native secondary structure will be tested to determine whether termination is structurally based. Finally, preliminary observations in our laboratory suggest that the p51 subunit of reverse transcriptase originally thought to be inactive, does generate DNA on a RNA primed DNA template. Thus, mutagenized p51 will be mixed with native p66 to assess its role in generating the (+) strand DNA intermediate. These studies should serve to identify a key control point in the HIV life cycle and perhaps a potential target for therapeutic strategies.