Oxidative Base Damage of RNA and Retroviral Mutation

描述（由申请人提供）：氧化碱基损伤通过显著增加DNA复制过程中错对形成的频率来促进突变。虽然我们对基因组DNA复制过程中氧化碱基损伤如何导致突变了解甚多，但对RNA复制过程中氧化碱基损伤的影响了解甚少。氧化碱基损伤在逆转录病毒如HIV的高突变中可能是特别重要的。这是因为感染相关的氧化应激大大增加了核酸碱基损伤；RNA不容易修复；复制是由易出错的逆转录酶介导的。在这项资助的初始阶段，我们确定了一些氧化损伤在HIV逆转录酶（HIV- rt）复制过程中促进碱基替换的高频率。这一继续应用的主要焦点是进一步确定机制，即核酸的氧化碱基损伤有助于HIV高突变。我们将开发一种综合的机制方法，旨在研究影响HIV超突变的因素。动力学方法将用于检测逆转录病毒复制的RNA和DNA模板导向阶段氧化碱基的差异利用。RNA模板二级结构和序列背景对氧化碱基诱导的高突变的影响将被确定。参与复制的逆转录病毒辅助蛋白如何改变这些影响将被研究。由铁诱导的氧化应激、过氧自由基、一氧化氮和过氧亚硝酸盐引起的RNA碱基损伤对逆转录病毒突变谱的影响将通过一种新的逆转录病毒正向突变试验直接确定。氧化碱基损伤、易出错逆转录酶介导的逆转录病毒复制的高频率、模板结构和组成以及其他蛋白质因子共同促成了HIV的高突变现象。这导致病毒从免疫系统的监视中逃脱，并在艾滋病患者中迅速产生耐药性。确定可被操纵以减弱超突变的因素应允许制定旨在限制获得耐药性和提高常规抗病毒治疗有效性的策略。另外，提高超突变率的策略可能会增加缺陷病毒或无活力病毒的产生，从而限制活动性感染的程度。

DESCRIPTION (provided by applicant): Oxidative base damage promotes mutations by substantially increasing the frequency of mispair formation during DNA replication. While much is known about how oxidative base damage contributes to mutations during the replication of genomic DNA, much less is known about the impact of oxidative base damage for replication of RNA. Oxidative base damage is likely to be of particular importance in the hypermutation of retroviruses such as HIV. This is because infection associated oxidative stress substantially increases nucleic acid base damage; RNA is not subject to repair; and replication is mediated by error prone reverse transcriptase. In the initial period of funding for this grant, we established that several oxidative lesions promote base substitutions at high frequencies during replication by HIV reverse transcriptase (HIV-RT). A primary focus of this continuation application is to further define the mechanisms whereby oxidative base damage of nucleic acids contributes to HIV hypermutation. We will develop an integrated mechanistic approach aimed at investigating factors which influence hypermutation in HIV. Kinetic methods will be used to examine the differential utilization of oxidized bases during the RNA and DNA template directed phases of retroviral replication. The influence of RNA template secondary structure and sequence context on oxidized base induced hypermutation will be determined. How retroviral accessory proteins which participate in replication modify these effects will be studied. The influence on retroviral mutation spectra resulting from RNA base damage caused by iron induced oxidative stress, peroxyl radicals, nitric oxide and peroxynitrite will be directly ascertained using a novel retroviral forward mutation assay.Oxidative base damage, a high frequency of retroviral replication mediated by error prone reverse transcriptase, template structure and composition, and additional protein factors contribute synergistically to the phenomenon of hypermutation in HIV. This results in viral escape from immune system surveillance and the rapid evolution of drug resistance in people with AIDS. Identifying factors that can be manipulated to attenuate hypermutation should allow for the development of strategies aimed at limiting the acquisition of drug resistance and improve the effectiveness of conventional antiviral therapies. Alternatively, strategies to increase the rate of hypermutation could increase the production of defective or non-viable virus, thus limiting the extent of active infection.