BIOCHEMICAL DETERMINANTS OF RETROVIRAL MUTATION

人类免疫缺陷病毒(HIV)的高度遗传变异性 严重阻碍了有效抗病毒治疗的发展。 这种高度变异性是由逆转录病毒的高度不忠所驱动的 导致频繁突变和快速选择的复制 新的变种。许多突变导致宿主范围、组织发生变化 特异性、细胞致病性、免疫原性和/或药物敏感性 从而极大地影响疾病的进展。 病毒变异的重要性显而易见，然而，病毒变异的机制 人们对逆转录病毒突变知之甚少。在此之前，我们展示了 纯化的HIV逆转录酶(RT)在体外容易出错，因此 可能在产生新的病毒变种方面起到了作用。然而，更多 我们实验室最近的研究表明，RT的准确性 体外和体内在选择的基因座和纯化的基因座上存在显著差异 RT在体外复制HIV基因组序列的效率非常低。 总而言之，这些数据表明，利用纯化的RT进行保真度分析 在体外是过于简单的，并不能完全代表分子 在体内逆转录病毒复制和突变过程中发生的事件。 我们建议更好地描述逆转录病毒的生化基础。 通过比较细胞系统和无细胞系统中的突变来实现突变。为 这些研究我们将利用Moloney小鼠白血病病毒(MLV)为基础 在体外和体内都很容易处理的载体系统 复制和突变分析。MLV将作为逆转录病毒的模型 并将在今后的平行研究中酌情与艾滋病毒进行对比。 开发了艾滋病毒载体系统。具体目的是：1)比较 正向突变分析中的逆转录病毒突变率和RT保真度 对数百个独立基因座的基因突变进行评分；2) 确定病毒和细胞因素对RT保真度的影响 研究渗透性病毒粒子和直接复制的准确性 与培养病毒中产生的突变和催化的错误进行比较 通过在相同的遗传位点进行体外纯化RT；3)确定 体内和体外复制错误的链特异性 从而确定RNA模板和DNA模板的相对贡献 RT错误以及RNA聚合酶II对逆转录病毒的不忠 变种。 总而言之，这些研究将描述利率和首选类型 模型逆转录病毒中出现的突变，并将确定其作用 病毒和细胞因子在逆转录病毒突变中的作用。一个 了解逆转录病毒突变的机制是基本的 了解逆转录病毒的进化和它们的便利 以躲避免疫监控和抗病毒药物。此外，一个 逆转录病毒变异的生化基础的表征和 参与这些频繁事件的分子因素可能识别新的 扰乱逆转录病毒变异率从而延迟的策略 HIV抗药性或加速子代病毒突变自杀。

The high genetic variability of human immunodeficiency virus (HIV) has seriously impaired the development of efficacious antiviral therapy. This hypervariability is driven by the high infidelity of retroviral replication which results in frequent mutation and the rapid selection of new variants. Many mutations confer altered host range, tissue specificity, cytopathogenicity, immunogenicity and/or drug sensitivity to the progeny virus and thus significantly affect disease progression. The importance of virus variation is evident, however, the mechanisms of retroviral mutation are poorly understood. Previously, we showed that purified HIV reverse transcriptase (RT) is error-prone in vitro and thus likely plays a role in generating new virus variants. However, more recent studies in our laboratory revealed that the accuracy of RT in vitro and in vivo differs significantly at select loci and that purified RT copies HIV genomic sequences in vitro with very low efficiency. Together, these data suggest that fidelity assays utilizing purified RTs in vitro are overly simplistic and do not fully represent the molecular events occurring during retroviral replication and mutation in vivo. We propose to better characterize the biochemical basis of retroviral mutation by comparing mutagenesis in cellular and cell-free systems. For these studies we will utilize Moloney murine leukemia virus (MLV)-based vector systems that are highly tractable for both in vitro and in vivo replication and mutation assays. MLV will serve as a model retrovirus and will be contrasted to HIV in future parallel studies as appropriate HIV vector systems are developed. The specific aims are: 1) to compare retroviral mutation rates and RT fidelity in forward mutation assays that genetically score mutations at several hundred independent loci; 2) to determine the influence of viral and cellular factor on RT fidelity by studying replication accuracy in permeabilized virions and by direct comparisons with mutations arising in cultured virus and errors catalyzed by purified RT in vitro at identical genetic loci; and 3) to determine the strand-specificity of replication errors both in vivo and in vitro and thus determine the relative contributions of RNA- and DNA-templated RT errors as well as RNA polymerase II infidelity to retroviral variation. Together, these studies will characterize the rates and preferred types of mutations arising in a model retrovirus and will identify the roles of viral and cellular factors in retroviral mutagenesis. An understanding of the mechanisms of retroviral mutations is fundamental to an understanding of the evolution of retroviruses and their facility to escape immunosurveillance and antiviral drugs. Moreover, a characterization of the biochemical basis of retroviral mutation and the molecular factors involved in these frequent events may identify novel strategies for perturbing retroviral mutations rates and thereby delaying HIV drug resistance or accelerating mutational suicide of progeny virus.