REVERSE TRANSCRIPTASE TEMPLATE SWITCHING AND FIDELITY

描述（改编自申请人摘要）： 逆转录病毒的数量取决于突变和重组 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。 高突变率 逆转录病毒是由于病毒编码的逆转录酶， 缺乏校对功能，持续性差。 突变发生 通过错误掺入、错位诱变和分子内或 分子间链转换 模板转换是一个过程， 病毒复制所需的，可导致缺失，复制， 重组 以前的研究已经确定了突变率和 通过病毒复制可能发生的突变谱。 当前 应用程序旨在定义RT切换模板的机制， 逆转录酶的结构决定簇和逆转录复合物， 对忠诚很重要 提出了四个具体目标：1）利用 编码直接重复序列的逆转录病毒载体， RNA和DNA依赖性DNA中模板转换的相对速率 合成，以及直接重复序列之间的距离，RNA二聚体 连接信号和模板-引物氢键对模板转换的影响; 2)进行靶向和随机突变分析，以确定结构 逆转录酶和RNase H的特征对于 DNA合成的准确性。 3)确定环境因素，如 由于核苷酸库失衡和抗病毒药物影响逆转录病毒 突变率 4)探测反转录复合体的结构 通过确定DNA合成是否在共包装的RNA上启动， 负链转移是否由假定的环状结构促进 病毒RNA的。

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.