Biochemistry of HIV reverse transcriptase fidelity and inhibitor interactions

 描述(申请人提供)：由于病毒的快速变异和产生可绕过免疫反应和抵抗药物治疗的基因变异的能力，抗击艾滋病毒的努力受到了阻碍。艾滋病毒的巨大遗传多样性极大地增加了为一种病毒生产有效疫苗的艰巨任务，这种病毒能够通过细胞间的接触传播，并将其遗传物质隐藏在休眠的储存细胞群体中的前病毒状态。高效抗逆转录病毒疗法(HAART)一直是治疗艾滋病毒患者的有力工具，但它并没有有效地惠及最脆弱的人群，同时HAART中使用的许多药物不可避免地出现了耐药性。今后有必要采取其他办法来帮助控制和克服艾滋病毒感染和艾滋病。这项应用旨在通过更清楚地定义HIV逆转录酶(RT)保真度的机制并检测生理条件对保真度的影响以及常用RT抑制剂的使用来为这些目标做出贡献。显示RT生物化学如何在细胞条件下发生的实验表明，目前关于RT保真度以及与AZT和ddC等药物相互作用的文献具有误导性。拟议的实验结果将更清楚地定义RT在细胞中的工作方式，使开发和评估潜在的新药变得更容易。与罗格斯大学Eddy Arnold博士的团队合作，还展示了一种使用与RT结合非常紧密的引物-模板模拟物来结晶HIV RT的新方法。这种方法首次允许RT在没有交联剂的情况下快速结晶，并导致形成催化活性晶体，可以用RT抑制剂处理这些晶体来研究RT-抑制剂复合体的结构。这些信息可用于基于结构的新型RT抑制剂的设计。适配子(与目标蛋白质结合非常紧密的核酸)可能 可用于诊断和治疗应用，如替代生化分析中的抗体(例如，酶联免疫吸附试验)，用作生物传感器，用作研究病毒分子生物学的工具，以及作为抗病毒药物的模型。针对艾滋病毒和其他逆转录病毒的适体已被证明在细胞和动物模型中抑制病毒复制。在这项提案中，一类新的 由“Xeno”核酸(XNA)组成的HIV RT适配子将与用于上述结晶的新型引物-模板模拟适配子一起进行病毒抑制测试。XNAs由与正常核苷酸相似的核苷酸模拟物组成，但包含有别于正常核苷酸的独特的化学和结构修饰。XNA适配子是独一无二的，因为核苷的非自然结构不太容易受到降解酶和其他恶劣条件的影响，同时它们也不太可能被天然免疫系统识别，而不是目前的RNA和DNA适配子。

 DESCRIPTION (provided by applicant): Attempts to combat HIV have been hampered due to the virus's ability to rapidly mutate and produce genetic variants that can circumvent the immune response and resist drug therapy. The tremendous genetic diversity of HIV has greatly complicated the already daunting task of producing an effective vaccine for a virus capable of spreading through cell to cell contact and concealing its genetic material in the proviral state within populations of dormant reservoir cells. Highly Active Anti-Retroviral Therapy (HAART) has been a powerful tool for treating HIV patients but it has not effectively reached the most vulnerable populations, while resistance to many of the drugs used in HAART has inevitably emerged. Alternative approaches will be necessary in the future to help control and overcome HIV infections and AIDS. This application aims to contribute to these goals by more clearly defining the mechanism of HIV reverse transcriptase (RT) fidelity and examining the effect of physiological conditions on fidelity and the utilization of commonly used RT inhibitors. Experiments that show how RT biochemistry occurs under cellular conditions indicate that current literature on RT fidelity and interactions with drugs like AZT and ddC is misleading. Results from the proposed experiments will more clearly define how RT works in the cell making it easier to develop and evaluate potential new drugs. A novel approach for crystallization of HIV RT using primer-template mimics that bind very tightly to RT is also demonstrated in collaboration with Dr. Eddy Arnold's group at Rutgers. This method allows for the first time, rapid crystallization of RT in the absence of cross-linking and leads to the formation of catalytically active crystals that can be treated with RT inhibitors to investigate the structure o the RT-inhibitor complexes. Information can be used for structure-based design of novel RT inhibitors. Aptamers (nucleic acids that bind extremely tightly to target proteins) can potentially be used for diagnostic and therapeutic applications, such as replacements of antibodies in biochemical assays (e.g. ELISA), utilization as biosensors, as tools for studying virus molecular biology, and as models for antiviral drugs. Aptamers specific to HIV and other retroviruses have been shown to inhibit virus replication in cell and animal models. In this proposal, a new class of aptamers to HIV RT that are composed of "Xeno" nucleic acids (XNA) will be tested for viral inhibition along with the novel primer-template mimicking aptamers used for crystallization above. XNAs are composed of nucleotide mimics that resemble normal nucleotides but contain unique chemical and structural modification that differentiate them from normal nucleotides. XNA aptamers are unique because the unnatural structure of the nucleoside is less susceptible to degradative enzymes and other harsh conditions, while they are also less likely to be recognized by the innate immune system than current RNA and DNA aptamers.