DNA SYNTHESIS AND RECOMBINATION BY HIV DNA POLYMERASE

简介(摘自申请者摘要)：拟进行的研究主要集中在 HIV-1逆转录酶的聚合酶和核糖核酸酶H功能的研究 病毒复制和重组。在感染艾滋病毒的个人中，5至10 有百分比的HIV临床分离株被鉴定为重组。这 序列的重组有助于病毒适应。检视 利用纯化组分的重组机制，我们发现发夹 逆转录病毒基因组5‘端的结构，如HIV-1TAR， DNA通过模板交换促进同源重组 底漆。我们提出了一种机制，发夹相互作用可以带来 将RNA基因组的同源区域聚集在一起，促进交叉。近期 用MLV进行的细胞培养研究突出了亲吻环，一个很好的特征 逆转录病毒基因组二聚化结构域内的茎环结构，如 重组的热点。在接吻环中，环序列允许碱基 在两个完全相同的茎环副本之间配对。我们现在怀疑 在自然界中促进交叉的能力是有层次的 茎环，基于增强模板的特定结构特征 相互作用，并为底物转移创造有利的几何形状。我们建议 以确定如何通过相互作用的发夹来促进模板切换，以及 发夹的结构特征与相互作用稳定性是否相关 直接与交叉频率连接。改变茎和环的序列变化 将分析大小、互补性和接吻稳定性。 RT的RNaseH功能将基因组RNA切割成寡聚体 减去链合成。非PPT RNA寡聚体通过RT通过一种 从RNA 5‘端进行的一系列一次和二次切割的有序序列。从… 临床分离株中，我们发现了非核苷类耐药的RT突变体。 P237L，在这种RNA 5‘端定向切割模式下存在缺陷。我们 对5‘端切割的机理进行了分析，并进行了实验研究 确定这一过程中的缺陷会如何损害病毒复制。

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.