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。虽然人们对氧化碱基损伤如何导致基因组DNA复制过程中的突变知之甚少，但对氧化碱基损伤对RNA复制的影响却知之甚少。在艾滋病毒等逆转录病毒的超突变中，氧化碱基损伤可能特别重要。这是因为感染相关的氧化应激大大增加了核酸碱基的损伤；RNA不受修复；复制是由容易出错的逆转录酶介导的。在为这笔赠款提供资金的最初时期，我们确定了几种氧化损伤在艾滋病毒逆转录酶(HIV-RT)复制过程中促进了高频的碱基替换。这一继续应用的一个主要焦点是进一步确定核酸的氧化碱基损伤导致艾滋病毒超突变的机制。我们将开发一种综合机制方法，旨在调查影响艾滋病毒过度突变的因素。动力学方法将被用来检验在逆转录病毒复制的RNA和DNA模板导向阶段氧化碱基的不同利用。将确定RNA模板、二级结构和序列上下文对氧化碱基诱导的超突变的影响。参与复制的逆转录病毒辅助蛋白如何改变这些效应将被研究。利用一种新的逆转录病毒正向突变分析方法，可以直接确定由铁诱导的氧化应激、过氧基、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。氧化性碱基损伤、容易出错的逆转录酶、模板结构和组成以及额外的蛋白质因素协同作用导致HIV的超突变现象。这导致艾滋病患者的病毒逃脱了免疫系统的监控，并迅速产生了耐药性。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抗药性并提高传统抗病毒疗法有效性的战略。或者，提高高度突变率的策略可能会增加缺陷或不能存活的病毒的产量，从而限制活跃感染的程度。

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.