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]

最近，针对病毒进入和成熟的新药已被添加到不断增长的抗病毒药物库中，旨在阻止艾滋病毒感染的持续传播。此外，参与艾滋病毒复制关键步骤的宿主因素正在考虑作为治疗干预的目标。尽管有这些有前景的方法，病毒酶、蛋白酶 (PR)、逆转录酶 (RT) 和整合酶 (IN) 仍然是高效抗逆转录病毒治疗的主要目标，近 20 种药物用于对抗 PR 和 RT，IN 抑制剂正在进行临床研究。试验。关于 HIV-1 RT，我们的部分将生物化学和生物物理学与结构生物学、分子生物学和化学生物学相结合，提供其在将入侵病毒的 RNA 转化为具有整合能力的双链 DNA 中的作用的分子细节。使用核苷和氨基酸类似物为代表复制中间体的复合物结构提供新的敏锐性，补充了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补充，同时引入单分子荧光研究来探测各种核酸双链体上的 RT 方向。在本项目中，我们研究了 (+) 链 DNA 合成的多嘌呤束 (PPT) 引物的结构特征，介导其被 HIV-1 RT 的 RNase H 结构域识别。这些研究表明，模板核碱基可以被去除，而不会损失易断裂的-1/+1磷酸二酯键的切割特异性，而RNA引物中的等效损伤是抑制性的。随后的研究确定了 PPT 引物中对其功能至关重要的各个碱基。嘌呤类似物的使用还使我们能够确定嘌呤环的环外基团在这些关键位置的作用。最后，使用类似物取代的底物和 HIV-1 RT 突变体的组合，我们使用核苷类似物来了解单链模板如何在进入 HIV-1 DNA 聚合酶活性位点之前与 Finger 子结构域的残基相互作用RT。 我们与 NCI/SAIC 蛋白质表达实验室合作，将无细胞翻译系统与新型抑制 tRNA 技术结合起来，将非天然氨基酸类似物定点插入 p66/p51 HIV-1 RT 的 p66 亚基中（Sitaraman 等人） .，2003）。使用这种方法（Klarmann 等，2004），我们用间氟-Tyr 和去甲-Tyr 取代 DNA 聚合酶 -Tyr-Met-Asp-Asp- 活性位点基序的 Tyr183，后者导致丢失RNA 依赖性 DNA 聚合酶活性的降低，而 DNA 依赖性 DNA 聚合酶活性不受影响。在随后的研究中，我们评估了 5 个 HIV-1 RT 变体，其 p66 亚基的 115 位含有酪氨酸类似物。每个突变体都保留了显着的 DNA 聚合酶活性，并选择了两个进行详细的动力学分析。与野生型 RT 相比，氨甲基-Phe115 RT 更有效地掺入 dCTP，并且对链终止核苷类似物 (-)-β-2,3-二脱氧-3-硫胞苷三磷酸 (3TCTP) 具有抗性。 2-萘基-Tyr115 RT 活性在低 dCTP 浓度下显着受损，并且所有测试的 dCTP 类似物的动力学速度都较慢。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所观察到的催化速率变化的可能机制。最后，基因操纵的大肠杆菌菌株已被用于位点特异性地引入可光活化的氨基酸，以与各种生物分子交联。 这项工作的成果是，于 2009 年提交了一份关于“荧光团非共价附着于寡核苷酸以进行高通量测序”的员工发明报告（发明人 Jason W. Rauch 和 Stuart J. Le Grice）。 [对应于 Le Grice 项目1 2007 年 4 月 HIV 耐药项目实地考察报告]