Oxidative Base Damage of RNA and Retroviral Mutation

RNA 氧化碱基损伤和逆转录病毒突变

• 批准号: 6775643
• 负责人: JOHN S TERMINI
• 金额: $ 25.38万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6775643
• 关键词: DNA damage; RNA directed DNA polymerase; Retroviridae; flow cytometry; free radical oxygen; gene mutation; human immunodeficiency virus 1; nucleic acid biosynthesis; nucleic acid sequence; nucleic acid structure; nucleocapsid; oxidation; transfection; virus DNA; virus RNA; virus genetics; virus protein

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.

描述（由申请人提供）：氧化性碱基损伤通过显著增加DNA复制期间错配形成的频率来促进突变。虽然人们对氧化性碱基损伤在基因组DNA复制过程中如何导致突变了解得很多，但对氧化性碱基损伤对RNA复制的影响知之甚少。氧化性碱基损伤在逆转录病毒如HIV的超突变中可能特别重要。这是因为感染相关的氧化应激大大增加了核酸碱基损伤; RNA不受修复;复制由易错逆转录酶介导。在该资助的最初阶段，我们确定了几种氧化损伤在HIV逆转录酶（HIV-RT）复制过程中以高频率促进碱基替换。该继续申请的主要焦点是进一步定义核酸的氧化碱基损伤促成HIV超突变的机制。我们将开发一个综合的机制的方法，旨在调查影响HIV超突变的因素。动力学方法将用于检查在逆转录病毒复制的RNA和DNA模板导向阶段期间氧化碱基的差异利用。将确定RNA模板二级结构和序列背景对氧化碱基诱导的超突变的影响。将研究参与复制的逆转录病毒辅助蛋白如何改变这些效应。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使用新的逆转录病毒正向突变测定法直接确定。氧化碱基损伤，由易错逆转录酶介导的高频率逆转录病毒复制，模板结构和组成，和其他蛋白质因子协同地促成HIV中的超突变现象。这导致病毒逃避免疫系统的监视，并在艾滋病患者中迅速进化出耐药性。识别可以被操纵以减弱超突变的因素应该允许旨在限制获得耐药性和提高常规抗病毒治疗的有效性的策略的发展。或者，增加超突变率的策略可以增加缺陷或非存活病毒的产生，从而限制活动性感染的程度。