DNA SYNTHESIS AND RECOMBINATION BY HIV DNA POLYMERASE

描述（改编自申请人摘要）：拟定研究的重点是 HIV-1逆转录酶（RT）的聚合酶和RNase H功能 病毒复制和重组。在艾滋病毒感染者中，5至10 %的HIV临床分离株被鉴定为重组体。这 序列的重组有助于病毒适应。检查 使用纯化的组分的重组机制，我们发现发夹 逆转录病毒基因组5 '端的结构，如HIV-1 TAR， 通过DNA的模板交换促进同源重组 引物我们提出了一种机制，通过这种机制，发夹相互作用可以带来 RNA基因组的同源区域在一起，促进交叉。最近 使用MLV进行的细胞培养研究突出了吻环， 逆转录病毒基因组的二聚化结构域内的茎环结构，如 重组的热点在吻环中，环序列允许碱基 两个相同的茎环之间的配对。我们现在怀疑 在自然界中促进跨界的能力是有等级的 茎环，基于增强模板的特定结构特征 这是一个良好的相互作用，并为引物转移创造有利的几何形状。我们提出 以确定相互作用的发夹如何促进模板开关， 发夹的结构特征和相互作用稳定性是否相关 与分频器频率直接相关。改变茎和环的序列变化 将分析大小、互补性和吻接稳定性。 RT的RNase H功能将基因组RNA切割成寡聚体， 负链合成。非PPT RNA寡聚体通过逆转录酶降解， 从RNA 5 '端进行的有序的一级和二级切割。从 在临床分离株中，我们发现了非核苷类药物耐药的RT突变体，例如， P237 L，其在RNA 5 '端定向切割的这种模式中有缺陷。我们 提出了5 '-末端切割机制的分析，并进行了实验， 确定这个过程中的缺陷如何损害病毒复制。

Description (Adapted from Applicant's abstract): The proposed studies focus on the polymerase and RNase H functions of HIV-1 reverse transcriptase (RT) in viral replication and recombination. In HIV infected individuals, 5 to 10 percent of HIV clinical isolates are identified as recombinant. This recombining of sequences contributes to viral adaptation. Examining recombination mechanisms using purified components, we found that the hairpin structures at the 5'-ends of retroviral genomes, such as the HIV-1 TAR, facilitate homologous recombination through template exchange by the DNA primer. We have proposed a mechanism whereby hairpin interactions can bring homologous regions of the RNA genomes together, promoting crossovers. Recent cell culture studies using MLV highlight the kissing loop, a well characterized stem loop structure within the dimerization domain of the retroviral genome, as a hot spot for recombination. In kissing loops, the loop sequence allows base pairing between two identical copies of the stem loops. We now suspect that there is a hierarchy in the ability to promote crossovers in natural stem-loops, based on specific structural features that enhance template interactions, and create a favorable geometry for primer transfer. We propose to determine how template switches are facilitated by interacting hairpins, and whether structural features of the hairpin and interaction stability correlate directly with crossover frequency. Changes in sequence that alter stem and loop size, complementarity, and kissing stability will be analyzed. The RNase H function of RT cuts the genomic RNA into oligomers in the course of minus strand synthesis. Non-PPT RNA oligomers are degraded by RT through an ordered series of primary and secondary cuts made from the RNA 5'-end. From clinical isolates, we found non-nucleoside drug resistant RT mutants, e.g. P237L, that are defective in this mode of RNA 5'-end directed cleavage. We propose an analysis of the mechanism of 5'-end cleavage, and experiments to determine how defects in this process could impair viral replication.