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

HIV-1 cPP 束与 RT 抑制剂敏感性之间的机制相互作用

• 批准号: 8129154
• 负责人: Waaqo Boru Daddacha
• 金额: $ 4.18万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8129154
• 关键词: Affect; Affinity; Binding; Biochemical; CD4 Positive T Lymphocytes; Catalysis; Cells; Cellular Structures; Complex; DNA; DNA Structure; DNA biosynthesis; DNA-Directed DNA Polymerase; Defect; Dimerization; Doctor of Philosophy; Drug Delivery Systems; Drug resistance; Elements; Enzymes; Evolution; Excision; Genome; HIV; HIV-1; Highly Active Antiretroviral Therapy; In Vitro; Infection; Integrase; Interphase Cell; Kinetics; Laboratories; Microglia; Mutagenesis; Mutation; Nature; Nucleotides; Patients; Pharmaceutical Preparations; Play; Polymerase; Protease Inhibitor; Proteins; RNA; RNA-Directed DNA Polymerase; Reporting; Resistance; Reverse Transcriptase Inhibitors; Role; SIV; Specificity; Staging; Structural Models; Structure; Subfamily lentivirinae; Surgical Flaps; System; Testing; Time; Tropism; Ursidae Family; Variant; Viral; Viral Genome; Virus; Virus Diseases; Zidovudine; Zidovudine resistance; cell type; combat; interest; macrophage; mutant; non-nucleoside reverse transcriptase inhibitors; novel; nucleocytoplasmic transport; nucleoside analog; prevent; research study; resistant strain; simian human immunodeficiency virus; transduction efficiency; vector; viral DNA; viral RNA; viral resistance

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.

描述（由申请人提供）：以前我们的实验室已经证明，中央多嘌呤段（cPPT）的存在通过促进前病毒DNA合成，特别是在含有低细胞dNTP浓度的非分裂细胞中，增强了逆转录酶（RT）缺陷的HIV-1的转导效率。本申请中提出的实验集中于测试我们的假设：1）如果cPPT的不存在延迟了病毒复制动力学，则缺乏cPPT的HIV突变体变得对RT抑制剂如已知延迟病毒复制动力学的非核苷RT抑制剂更敏感。HIV-1复制动力学依赖于各种因素，这些因素也影响DNA合成的生化动力学，例如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。这解释了cPPT作为（+）DNA合成的额外引物如何加速前病毒DNA合成，特别是当DNA复制受到有限细胞dNTP池或酶促受损RT突变体的动力学阻碍时。由于众所周知NNRTI直接结合RT并延迟DNA合成，我们预测cPPT的去除可能会延迟DNA合成，降低病毒完成前病毒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及其载体系统进行检验。我们还预计，这种预测的NNRTI敏感性升高可以被细胞dNTP浓度的升高所抵消，这可以加速前病毒DNA合成并可能补偿cPPT缺陷。为了测试这一点，我们将采用dN处理来提高具有低dNTP浓度的细胞中的细胞dNTP库。2）cPPT的去除可能进一步延迟AZT抗性突变体病毒在AZT存在下的复制，最终使AZT抗性突变体对AZT重新敏感。HIV对AZT的耐药机制是独特的，与病毒对NNRTI和蛋白酶抑制剂的耐药相比，NNRTI和蛋白酶抑制剂阻止药物与病毒酶结合。HIV- 1通过从聚合DNA的3'末端去除掺入的AZTMP而产生AZT抗性。合乎逻辑的假设是，每当耐药RT分子去除AZTMP时，DNA合成暂停，最终延迟整体复制动力学。因此，在这项研究中，我们预测cPPT去除将通过延迟前病毒DNA合成的完成而使AZT抗性HIV突变体对AZT重新敏感。我们将使用携带抗AZT突变的HIV载体进行测试。3)我们的结构模型预测，A114驻留的HIV-1 RT是重要的dNTP结合亲和力，我们将测试这一假设，通过使用生物化学和动力学方法。由于预期这些突变体显示延迟的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，在前病毒DNA合成动力学中cPPT和dNTP结合亲和力之间是否存在任何机制性相互作用。 公共卫生关系：开发有效的高效抗逆转录病毒疗法（HAART）来对抗HIV-1是一个持续的挑战，这是由于病毒的快速进化和持续选择耐药菌株的诱变。HIV-1基因组的中央多嘌呤段（cPPT）序列在HIV-1整合酶的二聚化区域附近编码，对于启动HIV-1的（+）链前病毒合成至关重要[54]。理想情况下，如果整合酶二聚化区域被药物靶向，则病毒将被迫改变cPPT序列以变得对药物具有抗性。我们假设，如果整合酶的cPPT编码区被改变，它将破坏cPPT功能，使病毒对现有的HAART更敏感。我们的研究检查了这种假定的和新的HIV-1药物靶点。