Mechanistic Interplays between cPP Tract and RT Inhibitor Sensitivity of HIV-1

描述(申请人提供)：以前我们的实验室已经证明，中心多尿路(CPPT)的存在通过促进前病毒DNA的合成，特别是在含有低细胞dNTP浓度的未分裂细胞中，提高了带有缺陷逆转录酶(RT)的HIV-1的转导效率。本申请中提出的实验重点是验证我们的假设：1)如果cPPT的缺失延迟了病毒复制动力学，那么缺乏cPPT的HIV突变体对RT抑制剂(如非核苷类RT抑制剂)变得更加敏感，已知这些抑制剂可以推迟病毒复制动力学。HIV-1复制动力学依赖于多种因素，这些因素也影响DNA合成的生化动力学，如dNTP底物浓度和RT蛋白的催化活性。另一种有效完成9.6kb DS前病毒DNA的方法是缩短模板的大小。这就解释了cPPT作为DNA合成的额外引物是如何加速前病毒DNA合成的，特别是当DNA复制受到有限的细胞dNTP池或酶活性受损的RT突变体的动力学阻碍时。由于众所周知，NNRTIs直接结合RT并延迟DNA合成，我们预测cPPT的去除可能延迟DNA合成，减少病毒完成前病毒DNA合成的机会，最终增加HIV对NNRTI的敏感性。这一假设将使用体外HIV-1及其载体系统进行验证。我们还预计，这种预测的NNRTI敏感性的升高可以被细胞内dNTP浓度的增加所抵消，dNTP浓度的增加可以加速前病毒DNA的合成，并可能弥补cPPT的缺陷。为了测试这一点，我们将采用DN处理来提高低dNTP浓度细胞中的dNTP池，2)移除cPPT可能会进一步推迟在AZT存在的情况下耐AZT突变病毒的复制，最终使AZT耐药突变株对AZT重新敏感。与病毒对NNRTI和蛋白酶抑制剂的耐药性相比，HIV对AZT的耐药性是独特的，后者阻止药物与病毒酶结合。HIV-1通过从聚合DNA的3‘端去除结合的AZTMP来产生对AZT的抗性。合乎逻辑的假设是，每当耐药RT分子移除AZTMP时，DNA合成就会暂停，最终推迟整体复制动力学。因此，在这项研究中，我们预测去除cPPT将通过延迟AZT存在下前病毒DNA合成的完成而使耐AZT的HIV突变体对AZT重新敏感。我们将使用携带AZT耐药突变的HIV载体进行测试。3)我们的结构模型预测HIV-1 RT的A114位对dNTP结合亲和力是重要的，我们将使用生化和动力学方法验证这一假设。由于这些突变体预计会表现出延迟的DNA合成动力学，特别是在低dNTP浓度下，通过使用这些HIV-1 RT突变体和cPPT突变，我们将测试在含有高和低细胞dNTP浓度的细胞类型中，cPPT和dNTP结合亲和力之间是否存在任何机制上的相互作用。 与公共卫生相关：开发有效、高效的抗逆转录病毒疗法(HAART)来对抗艾滋病毒-1是一项持续的挑战，因为病毒的快速进化和持续选择抗药性毒株的突变。HIV-1基因组的中央多尿路(CPPT)序列编码在HIV-1整合酶二聚区附近，对于启动HIV-1的链前合成是必不可少的[54]。理想情况下，如果整合酶二聚化区域是药物的靶点，病毒将被迫改变cPPT序列，以便对药物产生抗药性。我们推测，如果整合酶的cPPT编码区被改变，它将扰乱cPPT功能，使病毒对现有的HAART更加敏感。我们的研究检验了这个假定的和新的HIV-1药物靶点。

DESCRIPTION (provided by applicant): Previously our lab has shown that the presence of central polypurine tract (cPPT) enhances transduction efficiency of HIV-1 with defective reverse transcriptase (RT) by promoting the proviral DNA synthesis, particularly in non-dividing cells containing low cellular dNTP concentrations. The experiments proposed in this application focus on testing our hypothesis that: 1) if the absence of cPPT delays the viral replication kinetics, then HIV mutant lacking cPPT becomes more sensitive to RT inhibitors such as non-nucleoside RT inhibitors, known to delay the viral replication kinetics. HIV-1 replication kinetics relies on various elements that also affect the biochemical kinetics of DNA synthesis, such as dNTP substrate concentration and catalytic activity of RT protein. Another way to complete the 9.6 kb ds proviral DNA efficiently is to shorten the size of the template. This explains how cPPT, which serves as an additional primer for the (+) DNA synthesis, accelerates the proviral DNA synthesis especially when the DNA replication was kinetically hindered by either limited cellular dNTP pools or enzymatically compromised RT mutants. Since it is well known that NNRTIs binds RT directly and delay the DNA synthesis, we predict that the cPPT removal, which may delay the DNA synthesis, reduce the chance of the virus to complete proviral DNA synthesis, ultimately increasing the sensitivity of HIV to NNRTI. This hypothesis will be tested using in vitro HIV-1 and its vector systems. We also expect that this predicted elevated NNRTI sensitivity can be counteracted by the elevation of cellular dNTP concentration which can accelerate the proviral DNA synthesis and may compensate the cPPT defect. To test this we will employ a dN treatment to elevate cellular dNTP pool in cells with low dNTP concentration, 2) removal of cPPT may further delay the replication of AZT resistant mutant viruses in the presence of AZT, ultimately re- sensitizing the AZT resistant mutants to AZT. The mechanism of HIV resistance to AZT is unique, compare to viral resistance to NNRTI and protease inhibitors, which prevent drugs from binding to the viral enzymes. HIV- 1 renders AZT resistance by removing the incorporated AZTMP from the 3' end of the polymerizing DNA. It is logical to assume that whenever the drug resistant RT molecules remove AZTMP, the DNA synthesis pauses, ultimately delaying the overall replication kinetics. Thus, in this study, we predict that cPPT removal will re- sensitize the AZT resistant HIV mutant to AZT by delaying the completion of proviral DNA synthesis in the presence of AZT. We will test this using HIV vectors harboring AZT resistant mutation. 3) Our structural model predicts that the A114 reside of HIV-1 RT is important for the dNTP binding affinity, and we will test this hypothesis by using biochemical and kinetic approaches. Since these mutants are expected to display delayed DNA synthesis kinetics, especially at low dNTP concentrations, by employing these HIV-1 RT mutants and the cPPT mutations, we will test if there is any mechanistic interplay between cPPT and dNTP binding affinity in the proviral DNA synthesis kinetics in cell types containing high and low cellular dNTP concentration. PUBLIC HEALTH RELEVANCE: Developing effective highly active anti-retroviral therapy (HAART) to combat HIV-1 is a constant challenge due to fast viral evolution and mutagenesis that persistently selects drug resistant strains. The central polypurine tract (cPPT) sequence of the HIV-1 genome, which is encoded near the dimerization region of HIV-1 integrase, is essential for the initiation of the (+) strand proviral synthesis of HIV-1 [54]. Ideally, if the integrase dimerization region is targeted by a drug, the virus will be forced to alter the cPPT sequence in order to become resistant to the drug. We hypothesize that if the cPPT encoding region of integrase is altered, it will disrupt cPPT function making the virus more sensitive to existing HAART. Our studies examine this putative and novel HIV-1 drug target.