STRUCTURE-FUNCTION RELATION IN HIV REVERSE TRANSCRIPTION

逆转录酶是许多致癌基因的组成部分， 病毒，包括人类白血病和艾滋病病毒， 是病毒复制所必需的 长期目标， 建议是理解和定义生物化学， 逆转录酶的酶学和结构性质 这样逆转录的分子机制 予以澄清。 因此，本研究的主要目的是 鉴定和定义逆转录酶的结构域 其参与底物dNTPs和模板的结合， 引物以及那些负责表达反向- 转录酶相关核糖核酸酶H。 结果预计 阐明DNA酶促合成的基本机制 由逆转录酶催化，并可能提供一个深入了解 独特的性能/结构，这可能有助于设计 潜在的治疗剂。 我们分析了抗逆转录酶活性的机制 许多抑制剂，并确定了一些试剂， 似乎与逆转录酶反应， 方式 此外，我们还制定了标准化的方案， 将底物、模板引物和特异性试剂连接到 酶蛋白 这些试剂在建立 RT的位点特异性共价修饰， 允许标记、鉴定和分离携带 这些网站。 通过这种方式，我们希望识别域 负责MuLV和HIV的聚合酶和RNA酶H活性 逆转录酶 这些知识将非常有用， 了解许多抗病毒药物的确切机制（包括 抗艾滋病）药物。

Reverse transcriptase is an integral part of many oncogenic viruses, including those from human leukemia and AIDS virus, which is essential for viral replication. Long range goals of this proposal are to understand and define the biochemical, enzymological and structural properties of reverse transcriptase so that molecular mechanisms underlying reverse transcription could be clarified. The major objective, therefore, of this research is to identify and define structural domains of reverse transcriptase which are involved in the binding of substrate dNTPs and template- primer as well as those responsible for the expression of reverse- transcriptase associated Ribonuclease H. The results are expected to clarify the basic mechanisms of enzymatic synthesis of DNA catalyzed by reverse transcriptase and may provide an insight into unique properties/structures which may help in designing the potential therapeutic agents. We have analyzed mechanism of anti-reverse transcriptase activity of a number of inhibitors and have identified some reagents which appear to react with reverse transcriptase in a site-specific manner. In addition, we have standardized protocols for covalently linking substrates, template-primers and specific reagents to the enzyme protein. These reagents are quite useful in establishment of site-specific covalent modification of RT which in turn will permit labeling, identification and isolation of peptides carrying these sites. In this manner, we hope to identify domains responsible for the polymerase and RNAse H activity of MuLV and HIV reverse transcriptase. This knowledge will be very useful in understanding of precise mechanisms of many anti-viral (including anti-AIDS) drugs with presumptive anti-RT activity.