Nucleoside and Amino Acid Analogs as Probes of HIV Replication Complexes

核苷和氨基酸类似物作为 HIV 复制复合物的探针

• 批准号: 8157323
• 负责人: Stuart F. J. Le Grice
• 金额: $ 71.62万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157323
• 关键词: Amino Acids; Complex; HIV; Nucleosides; analog

Novel drugs, targeting virus entry and maturation, have recently been added to the growing armory of antiviral agents aimed at stemming the continuing spread of HIV infection. In addition, host factors participating in key steps in HIV replication are under consideration as targets for therapeutic intervention. Despite these promising approaches, the viral enzymes, protease (PR), reverse transcriptase (RT) and integrase (IN), remain primary targets of highly active antiretroviral therapy, with almost 20 drugs in use against PR and RT, and IN inhibitors undergoing clinical trials. With respect to HIV-1 RT, our section combines biochemistry and biophysics with structural, molecular, and chemical biology to provide molecular details of its role in converting RNA of the invading virus into integration-competent double-stranded DNA. Using nucleoside and amino acid analogs to provide novel acumen into the structures of complexes representing replication intermediates complements traditional nucleic acid and protein mutagenesis methods. Chemical nucleic acid footprinting is now complemented with direct mass spectrometric analysis, while single-molecule fluorescence studies have been introduced to probe the orientation of RT on a variety of nucleic acid duplexes. In this project, we have investigated structural features of the polypurine tract (PPT) primer of (+) strand DNA synthesis mediating its recognition by the RNase H domain of HIV-1 RT. Such studies indicated that template nucleobases can be removed without loss of cleavage specificity at the scissile -1/+1 phosphodiester bond, while equivalent lesions in the RNA primer are inhibitory. Subsequent studies identified individual bases of the PPT primer that are critical to its function. The use of purine analogs has also allowed us to determine the role of the exocyclic groups of the purine ring at these critical positions. Finally, using a combination of analog-substituted substrates and mutants of HIV-1 RT, we have used nucleoside analogs to understand how the single-stranded template interacts with residues of the fingers subdomain prior to access the DNA polymerase active site of HIV-1 RT. In collaboration with the NCI/SAIC Protein Expression Laboratory, we coupled a cell-free translation system with a novel suppressor tRNA technology to site-specifically insert unnatural amino acid analogs into the p66 subunit of p66/p51 HIV-1 RT (Sitaraman et al., 2003). Using this approach (Klarmann et al., 2004), we substituted m-fluoro-Tyr and nor-Tyr for Tyr183 of the DNA polymerase -Tyr-Met-Asp-Asp- active site motif, the latter of which resulted in the loss of RNA-dependent DNA polymerase activity while DNA-dependent DNA polymerase activity was unaffected. In a subsequent study, we evaluated five HIV-1 RT variants containing tyrosine analogs at position 115 of their p66 subunit. Each mutant retained significant DNA polymerase activity, and two were selected for detailed kinetic analysis. Aminomethyl-Phe115 RT incorporated dCTP more efficiently compared with wild-type RT and was resistant to the chain-terminating nucleoside analog (-)-beta-2,3dideoxy-3-thiacytidine triphosphate (3TCTP). 2-Naphthyl-Tyr115 RT activity was significantly impaired at low dCTP concentrations and was kinetically slower with all dCTP analogs tested. Structural models of HIV-1 RT ternary complexes containing these amino acid substitutions have revealed probable mechanisms of the observed catalytic rate changes. Finally, genetically manipulated E. coli strains have been used to site-specifically introduce photoactivable amino acids for crosslinking to a variety of biomolecules. As a result of this work, an Employee Invention Report on "Non-covalent attachment of fluorophores to oligonucleotides for high throughput sequencing" (Inventors Jason W. Rauch and Stuart J. Le Grice) was submitted in 2009. [Corresponds to Le Grice Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]

靶向病毒进入和成熟的新型药物最近被添加到旨在阻止HIV感染持续扩散的抗病毒剂的抗病毒药物中。此外，参与HIV复制中关键步骤的宿主因素正在考虑作为治疗干预的目标。尽管采用了这些有希望的方法，但病毒酶，蛋白酶（PR），逆转录酶（RT）和整合酶（IN）仍然是高度活性抗逆转录病毒疗法的主要靶标，对PR和RT使用了近20种药物，以及在接受临床试验的抑制剂中。关于HIV-1 RT，我们的部分将生物化学和生物物理学与结构，分子和化学生物学相结合，以提供其在将入侵病毒转化为综合性双链DNA中的RNA中作用的分子细节。使用核苷和氨基酸类似物，将新型敏锐度提供为代表复制中间体的复合物的结构，可以补充传统的核酸和蛋白质诱变方法。化学核酸足迹现在与直接质谱分析相辅相成，而已经引入了单分子荧光研究，以探测RT在多种核酸双工中的方向。在这个项目中，我们研究了（+）链DNA合成的息肉素道（PPT）底漆的结构特征，从而介导了HIV-1 RT的RNase H结构域的识别。这样的研究表明，可以在无剪cissile -1/+1磷酸二酯键的情况下去除模板核苷酸酶，而RNA底漆中的等效病变是抑制性的。随后的研究确定了对其功能至关重要的PPT引物的单个碱基。嘌呤类似物的使用还使我们能够确定嘌呤环在这些关键位置上的作用。最后，使用HIV-1 RT的模拟取代底物和突变体的组合，我们已经使用了核苷类似物来了解单链模板在访问HIV-1 RT的DNA聚合酶活性位点之前如何与手指子域的残基相互作用。 与NCI/SAIC蛋白表达实验室合作，我们将无细胞的翻译系统与一种新型的抑制器TRNA技术耦合在一起，以特异性地将非天然氨基酸类似物插入p66/p51 HIV-1 RT的p66亚基（Sitaraman等，2003）。使用这种方法（Klarmann等，2004），我们将M-fluoro-Tyr和Nor-TYR取代了DNA聚合酶-TYR-MET-MET-ASP-ASP-Active位点的Tyr183，后者的后者导致RNA依赖性DNA聚合酶活性的丧失，而DNA依赖性DNA依赖性DNA依赖性DNA依赖性DNA Polymersase活性。在随后的研究中，我们评估了五个HIV-1 RT变体，这些变体在其P66亚基的位置115处含有酪氨酸类似物。每个突变体都保留了明显的DNA聚合酶活性，并选择了两种进行详细的动力学分析。与野生型RT相比，氨基甲基-PHE115 RT掺入了DCTP，并且对链末端的核苷类似物（ - ） - β-2,3Dideoxy-3-三维氧基三磷酸三磷酸（3TTCTP）具有抵抗力。 2-萘基-TYR115 RT活性在低DCTP浓度下显着损害，并且在所有DCTP类似物测试的情况下，动力学较慢。含有这些氨基酸取代的HIV-1 RT三元复合物的结构模型揭示了观察到的催化速率变化的可能机制。最后，遗传操纵的大肠杆菌菌株已被用于特异性地引入光活性氨基酸，以交联到各种生物分子。 这项工作的结果是，员工发明报告“荧光团非共价附着在寡核苷酸上进行高通量测序”（发明人Jason W. Rauch和Stuart J. Le Grice）于2009年提交。