DNA Synthesis and Recombination by HIV DNA Polymerase
HIV DNA 聚合酶的 DNA 合成和重组
基本信息
- 批准号:7662419
- 负责人:
- 金额:$ 36.96万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1992
- 资助国家:美国
- 起止时间:1992-12-01 至 2012-08-31
- 项目状态:已结题
- 来源:
- 关键词:AddressAnti-HIV AgentsBindingBiological AssayCatalysisCell Culture SystemCell Culture TechniquesCharacteristicsComplementary DNAComplexDNADNA FoldingDNA biosynthesisDNA-Directed DNA PolymeraseDefectDissociationDrug resistanceEmployee StrikesEnzymesEscherichia coliEventExerciseFrequenciesGaggingGenesGenetic RecombinationGenomeHIVHIV-1Hot SpotHybridsImmune responseIn VitroInheritedInvadedLengthLinkMeasuresModelingMovementMulti-Drug ResistanceNucleotidesParentsPathway interactionsPharmacotherapyPolymersPositioning AttributePrimer ExtensionProcessProteinsRNARNA FoldingRNA-Directed DNA PolymeraseReactionRelative (related person)Resistance developmentResolutionRibonuclease HRoleRunningSimulateSiteStructureSystemTestingTimeTransfer RNAViralVirusVirus DiseasesWorkbasedesigngenome sequencingin vitro Assayin vivomigrationmutantpolymerizationreconstitutionresearch studytherapeutic targettraitviral RNA
项目摘要
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.
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产生病毒多样性。这种多样性有助于病毒逃避宿主的免疫反应和药物治疗。可通过RNA模板之间的链转移发生双链化。该机制也用于复制途径中的负链强终止转移。转移涉及将生长的cDNA引物从原始供体RNA转移到第二受体RNA。我们的工作重建重组在体外与纯蛋白质,并在体内的细胞培养,解决机制,驱动链转移。转移起始于病毒编码的逆转录酶(RT)暂停的位点,使其能够使用其RNase H功能来集中供体模板中的切口。转移可以通过多步骤过程发生,其中受体模板侵入供体模板中缺口位点处的DNA。cDNA-受体杂合体扩散,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探讨。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。我们正在调查的证据表明,折叠有助于时间依赖性失活的cDNA末端转移,以及高效率取决于机制,完成转移之前,可以发生失活。新的研究结果表明,转移可以通过一种称为邻近的机制发生,这种机制不涉及最初的杂交体的传播。我们将评估传播机制与邻近机制的相对贡献。有证据表明,RT必须解离以进行转移,并且RT必须行使其独特的5'端定向RNA酶H活性。我们正在确定是否其中一个或两个功能是必不可少的转移。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长度的一半以上的重组交叉的位置和频率。我们最初通过gag基因的一部分对DIS的459 bp区域进行了测序。它揭示了一个引人注目的重组峰值,其中该区域三分之二的交换发生在约100个核苷酸内。值得注意的是,我们成功地概括了体外链转移试验中的热点,使我们能够确定其结构和机制基础。总的来说,我们的工作结果将澄清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。公共卫生相关性:HIV-1在感染者体内迅速进化其结构。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耐药性。该病毒有一种机制,它可以联合收割机,它继承了两个不同的病毒亲本不同的耐药性性状。这种机制可以产生具有增加的或多重耐药性的病毒。我们的研究结果将有助于我们了解和击败这种机制,使抗艾滋病药物可以更有效。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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ROBERT A BAMBARA其他文献
ROBERT A BAMBARA的其他文献
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{{ truncateString('ROBERT A BAMBARA', 18)}}的其他基金
REGULATING GENOME FIDELITY AND CANCER PROGRESSION
调节基因组保真度和癌症进展
- 批准号:
8637495 - 财政年份:2014
- 资助金额:
$ 36.96万 - 项目类别:
DNA Synthesis and Recombination by HIV DNA Polymerase
HIV DNA 聚合酶的 DNA 合成和重组
- 批准号:
7903104 - 财政年份:1992
- 资助金额:
$ 36.96万 - 项目类别:
DNA SYNTHESIS AND RECOMBINATION BY HIV DNA POLYMERASE
HIV DNA 聚合酶的 DNA 合成和重组
- 批准号:
6147667 - 财政年份:1992
- 资助金额:
$ 36.96万 - 项目类别:
DNA synthesis and recombination by HIV DNA Polymerase
HIV DNA 聚合酶的 DNA 合成和重组
- 批准号:
7209002 - 财政年份:1992
- 资助金额:
$ 36.96万 - 项目类别:
DNA synthesis and recombination by HIV DNA Polymerase
HIV DNA 聚合酶的 DNA 合成和重组
- 批准号:
6863726 - 财政年份:1992
- 资助金额:
$ 36.96万 - 项目类别:
DNA synthesis and recombination by HIV DNA Polymerase
HIV DNA 聚合酶的 DNA 合成和重组
- 批准号:
6796474 - 财政年份:1992
- 资助金额:
$ 36.96万 - 项目类别:
DNA Synthesis and Recombination by HIV DNA Polymerase
HIV DNA 聚合酶的 DNA 合成和重组
- 批准号:
8132392 - 财政年份:1992
- 资助金额:
$ 36.96万 - 项目类别:
DNA SYNTHESIS AND RECOMBINATION BY HIV DNA POLYMERASE
HIV DNA 聚合酶的 DNA 合成和重组
- 批准号:
6385820 - 财政年份:1992
- 资助金额:
$ 36.96万 - 项目类别:
DNA Synthesis and Recombination by HIV DNA Polymerase
HIV DNA 聚合酶的 DNA 合成和重组
- 批准号:
7552500 - 财政年份:1992
- 资助金额:
$ 36.96万 - 项目类别:
DNA SYNTHESIS AND RECOMBINATION BY HIV DNA POLYMERASE
HIV DNA 聚合酶的 DNA 合成和重组
- 批准号:
6519544 - 财政年份:1992
- 资助金额:
$ 36.96万 - 项目类别:
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