REVERSE TRANSCRIPTASE TEMPLATE SWITCHING AND FIDELITY

描述(改编自申请人的摘要)： 逆转录病毒种群依赖于突变和重组 速率/复制周期、复制速率/时间和选择性 存在于人口中的力量。基因的高突变率 逆转录病毒是由病毒编码的逆转录酶引起的，它 缺乏校对功能，加工效率低。突变会发生 通过错误结合、错位突变和分子内或 分子间链交换。模板切换，这是一个过程 病毒复制所需的，可能会导致删除、复制和 重组。以前的研究已经定义了突变率和 通过病毒复制可能产生的突变范围。海流 应用程序旨在定义RT切换模板和 RT和逆转录复合体的结构决定因素 对保真度很重要。提出了四个具体目标：1)利用 直接编码重复序列的逆转录病毒载体在体内确定 RNA和DNA依赖的DNA中模板切换的相对速率 RNA二聚体的合成以及直接重复序列之间距离的影响 连接信号，模板切换上的模板-底物氢键； 2)进行有针对性的随机突变分析，以定义结构 逆转录酶和核糖核酸酶H的特性 DNA合成的准确性。3)确定环境因素是否 由于核苷酸池失衡和抗病毒药物影响逆转录病毒 突变率。4)探索逆转录复合体的结构 通过确定DNA合成是否在共包装的RNA和 环状结构是否促进负链转移 病毒的核糖核酸。

DESCRIPTION (Adapted from the applicant's abstract): Genetic variation in the retroviral populations depend on the mutation and recombination rates/replication cycle, the rate of replication/time and the selective forces that exist on the population. The high mutational rate of retroviruses is due to the virally encoded reverse transcriptase, which lacks proofreading function and has low processivity. Mutations occur through misincorporation, dislocation mutagenesis, and intramolecular or intermolecular strand switching. The template switching, which is a process required for viral replication, can lead to deletions, duplications, and recombination. Previous studies has defined the mutation rates and the spectrum of mutations possible through viral replication. The current application aims at defining the mechanism that RT switches templates and the structural determinants of RT and the reverse transcription complex that are important for fidelity. Four specific aims are proposed: 1) Utilize retroviral vectors encoding directly repeated sequences to determine in vivo the relative rates of template switching in RNA and DNA- dependent DNA synthesis, and the effects of distance between direct repeats, RNA dimer linkage signal, and template-primer hydrogen bonding on template switching; 2) Perform targeted and random mutational analysis to define structural features of reverse transcriptase and RNase H that are important for accuracy of DNA synthesis. 3) Determine whether environmental factors such as nucleotide pool imbalances and anti-viral drugs affect retroviral mutation rates. 4) Probe the structure of the reverse transcription complex by determining whether DNA synthesis initiates on both co-packaged RNAs and whether minus-strand transfer is promoted by a putative circular structure of the viral RNAs.