DNA Synthesis and Recombination by HIV DNA Polymerase

描述（由申请人提供）：HIV-1利用其两个RNA基因组之间的频繁重组来创造病毒多样性。这种多样性有助于病毒逃避宿主免疫反应和药物治疗。重组可以通过RNA模板之间的链转移发生。这种机制也被用于复制途径中的负链强停止转移。转移包括将生长的cDNA引物从原始供体RNA转移到第二受体RNA。我们的工作在体外用纯蛋白质重组，在体内细胞培养，解决驱动链转移的机制。转移开始于病毒编码逆转录酶（RT）暂停的位点，允许它使用其RNase H功能来集中供体模板中的切割。转移可以通过一个多步骤的过程发生，在这个过程中，受体模板侵入供体模板中有缺口的DNA。cDNA-受体杂交扩散直到cDNA的3'末端区域完成转移。然而，转移机制的主要部分尚未被探索。体外负链转移模型反应表明，RNA和DNA折叠是转移效率的重要决定因素。我们正在研究的证据表明，折叠有助于cDNA末端的时间依赖性失活转移，而高效率取决于在失活发生之前完成转移的机制。新的结果表明，转移可以通过一种称为邻近的机制发生，这种机制不涉及初始杂交的传播。我们将评估扩散机制和接近机制的相对贡献。有证据表明，RT必须解离转移，并且RT必须行使其独特的5'端定向RNase H活性。我们正在确定其中一项或两项职能是否对转移至关重要。最后，我们开发了一种病毒细胞培养系统，该系统以25个核苷酸的分辨率测量超过一半HIV-1长度的重组交叉的位置和频率。我们首先从DIS中通过gag基因的一部分测序了一个459 bp的区域。它揭示了一个惊人的重组高峰，其中该区域三分之二的交叉发生在大约100个核苷酸内。值得注意的是，我们成功地再现了体外链转移实验中的热点，使我们能够确定其结构和机制基础。总之，我们的工作结果将阐明HIV-1中链转移的确切机制和要求。这是治疗靶向链转移作为干扰HIV-1感染手段的第一步。公共卫生相关性：HIV-1在感染者体内迅速演变其结构。这使得它可以逃避免疫反应，并对目前所有的药物治疗产生抗药性。这种病毒有一种机制，它可以结合从两种不同病毒亲本遗传的不同耐药性特征。这种机制可以产生具有增强或多重耐药性的病毒。我们的研究结果将帮助我们理解和克服这一机制，从而使抗艾滋病药物更加有效。

DESCRIPTION (provided by applicant): HIV-1 uses frequent recombination between its two RNA genomes to create viral diversity. This diversity helps the virus to escape host immune response and drug therapy. Recombination can occur by strand transfer between RNA templates. This mechanism is also employed for the minus strand strong stop transfer in the replication pathway. Transfer involves a shift of the growing cDNA primer from the original donor RNA to a second acceptor RNA. Our work reconstituting recombination in vitro with pure proteins, and in vivo in cell culture, addresses mechanisms that drive strand transfer. Transfers initiate at sites where the virally encoded reverse transcriptase (RT) pauses, allowing it to use its RNase H function to concentrate cuts in the donor template. Transfers can occur by a multi-step process in which acceptor template invades the DNA at the gapped site in the donor template. The cDNA-acceptor hybrid spreads until the 3' terminal region of the cDNA completes transfer. However, major parts of the transfer mechanism are unexplored. Minus strand transfer model reactions in vitro indicate that RNA and DNA folding is an important determinant of transfer efficiency. We are investigating evidence that folding contributes to a time-dependent inactivation of cDNA ends for transfer, and that high efficiency depends on mechanisms that complete transfer before inactivation can occur. New results indicate that transfers can occur by a mechanism called proximity that does not involve spreading of the initial hybrid. We will evaluate the relative contributions of the spreading versus proximity mechanisms. Evidence suggests that the RT is obligated to dissociate for transfers, and that the RT must exercise its unique 5' end-directed RNase H activity. We are determining whether either or both functions are essential for transfer. Lastly, we developed a viral cell culture system that measures the positions and frequencies of recombination crossovers over more than half of the length of HIV-1 at a resolution of 25 nucleotides. We initially sequenced a 459 bp region from DIS through part of the gag gene. It revealed a striking peak of recombination in which two-thirds of crossovers in the region occurred within about 100 nucleotides. Significantly, we successfully recapitulated the hot spot in strand transfer assays in vitro, allowing us to determine its structural and mechanistic basis. Overall, results of our work will clarify the exact mechanisms and requirements of strand transfer in HIV-1. This is a first step to therapeutic targeting of strand transfer as a means of interfering with HIV-1 infection.PUBLIC HEALTH RELEVANCE: HIV-1 rapidly evolves its structure in infected people. This allows it to escape immune response and to develop resistance to all current attempts at drug therapy. The virus has a mechanism whereby it can combine different drug resistance traits that it inherited from two different virus parents. This mechanism can produce viruses with increased or multi-drug resistance. Our results will help us to understand and defeat this mechanism so that anti-AIDS drugs can be more effective.