DNA Synthesis and Recombination by HIV DNA Polymerase

描述(申请人提供)：HIV-1使用其两个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。这一机制也适用于复制途径中的负链强停止转移。转移包括将生长的cdna引物从最初的供体RNA转移到第二个受体RNA。我们的工作是在体外用纯蛋白质重组，并在细胞培养中重组，解决了驱动链转移的机制。转移开始于病毒编码的逆转录酶(RT)暂停的位置，使其能够使用其RNaseH功能来集中切割供体模板。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间隙的位置的DNA。CDNA3‘-受体杂交体扩散到cDNA3’端区完成转移。然而，转移机制的主要部分还没有被探索。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。我们正在调查证据表明，折叠有助于对用于转移的cDNA端进行时间依赖性的失活，并且高效率依赖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接近的机制发生，这种机制不涉及初始杂交的传播。我们将评估扩散机制与邻近机制的相对贡献。有证据表明，RT有义务解离以进行转移，RT必须行使其独特的5‘端定向的RNaseH活性。我们正在确定是否其中一个功能或两个功能对于转移是必不可少的。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HIV-1超过一半长度的重组交叉的位置和频率。我们最初通过部分Gag基因对DIS的459个碱基区进行了测序。它揭示了一个惊人的重组高峰，该地区三分之二的交叉发生在大约100个核苷酸以内。值得注意的是，我们成功地概括了体外链转移分析中的热点，使我们能够确定其结构和机制基础。总体而言，我们的工作结果将阐明HIV-1链转移的确切机制和要求。这是将链转移作为干扰HIV-1感染的一种手段的治疗靶向的第一步。PUBLIC健康相关性：HIV-1在感染者中迅速进化其结构。这使得它能够逃避免疫反应，并对目前所有的药物治疗尝试产生抵抗力。这种病毒有一种机制，它可以结合从两个不同的病毒父母那里遗传来的不同的耐药性特征。这种机制可以产生抗药性增强或多重抗药性的病毒。我们的结果将有助于我们理解和击败这一机制，从而使抗艾滋病药物能够更有效。

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.