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: 8530252
• 负责人: Waaqo Boru Daddacha
• 金额: $ 2.54万
• 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 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8530252
• 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 Targeting; 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.

描述（由申请人提供）：之前我们的实验室已经证明，中央多嘌呤通道（cPPT）的存在通过促进原病毒DNA合成来提高具有缺陷逆转录酶（RT）的HIV-1的转导效率，特别是在含有低细胞dNTP浓度的非分裂细胞中。本应用程序中提出的实验重点是验证我们的假设：1)如果缺乏cPPT延迟病毒复制动力学，那么缺乏cPPT的HIV突变体对RT抑制剂（如已知延迟病毒复制动力学的非核苷类RT抑制剂）变得更加敏感。HIV-1复制动力学依赖于影响DNA合成生化动力学的各种因素，如dNTP底物浓度和RT蛋白的催化活性。另一种有效完成9.6 kb原病毒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复制受到有限的细胞dNTP池或酶受损的RT突变体的动力学阻碍时。由于已知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，从而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。这一假设将在体外HIV-1及其载体系统中得到验证。我们还预计，这种预测的NNRTI敏感性升高可以通过细胞dNTP浓度的升高来抵消，后者可以加速原病毒DNA的合成，并可能补偿cPPT缺陷。为了验证这一点，我们将在低dNTP浓度的细胞中使用dN处理来提高细胞dNTP池，2)去除cPPT可能进一步延迟AZT存在下AZT抗性突变病毒的复制，最终使AZT抗性突变体对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的耐药性机制是独特的，后者可阻止药物与病毒酶结合。HIV- 1通过从聚合DNA的3'端去除合并的AZTMP而产生AZT抗性。我们可以合理地假设，只要耐药RT分子去除AZTMP， DNA合成就会暂停，最终延缓整个复制动力学。因此，在本研究中，我们预测去除cPPT将通过延迟AZT存在下原病毒DNA合成的完成，使AZT耐药HIV突变体对AZT重新敏感。我们将使用携带AZT抗性突变的HIV载体进行测试。3)我们的结构模型预测了HIV-1 RT的A114驻留对dNTP结合亲和力的重要作用，我们将通过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突变体，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相互作用。