Nucleoside and Amino Acid Analogs as Probes of HIV Replication Complexes

针对病毒进入和成熟的新型药物最近被添加到不断增长的抗病毒药物军械库中，旨在阻止艾滋病毒感染的持续传播。此外，参与HIV复制关键步骤的宿主因子正在被考虑作为治疗干预的靶点。尽管有这些有希望的方法，病毒酶，蛋白酶（PR），逆转录酶（RT）和整合酶（IN），仍然是高活性抗逆转录病毒治疗的主要目标，有近20种药物用于对抗PR和RT，而IN抑制剂正在进行临床试验。关于HIV-1 RT，我们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的RNA转化为整合能力双链DNA中的作用的分子细节。利用核苷和氨基酸类似物对代表复制中间体的复合物结构提供新的敏锐度，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辅相成，而单分子荧光研究已经被引入来探测各种核酸双链上RT的取向。本项目是我们部门目前承担的四个项目之一，我们研究了（+）链DNA合成的多嘌呤通道（PPT）引物的结构特征，该引物介导HIV-1 rt的RNase H结构域对其的识别。这些研究表明，模板核碱基可以在可剪切的-1/+1磷酸二酯键上被去除而不会失去切割特异性，而RNA引物中的等效损伤是抑制的。随后的研究确定了PPT引物的各个碱基，这些碱基对其功能至关重要。嘌呤类似物的使用也使我们能够确定嘌呤环在这些关键位置的外环基团的作用。最后，使用类似物取代底物和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 RT的DNA聚合酶活性位点之前与手指亚结构域的残基相互作用。我们将无细胞翻译系统与一种新型的tRNA抑制技术结合起来，将非天然氨基酸类似物特异地插入到p66/p51 HIV-1 RT的p66亚基中（Sitaraman et al., 2003）。利用这种方法（Klarmann et al., 2004），我们用m-fluoro-Tyr和no - tyr取代了DNA聚合酶- tyr - met - asp - asp活性位点基序的Tyr183，后者导致rna依赖的DNA聚合酶活性丧失，而DNA依赖的DNA聚合酶活性不受影响。在随后的研究中，我们评估了5个在p66亚基115位含有酪氨酸类似物的HIV-1 RT变体。每个突变体都保留了显著的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，aminomemethyl - phe115 RT更有效地结合了dCTP，并且对链终止核苷类似物(-)- β -2,3dideoxy-3-thiacytidine triphosphate （3TCTP）具有抗性。2-萘酰基tyr115 RT活性在低dCTP浓度下显著受损，在所有dCTP类似物的测试中，RT活性都减慢。含有这些氨基酸取代的HIV-1 RT三元配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，基因操纵的大肠杆菌菌株已被用于位点特异性地引入光活化氨基酸，用于与各种生物分子交联。由于这项工作，2009年提交了一份关于“用于高通量测序的荧光团与寡核苷酸的非共价附着”的员工发明报告（发明者Jason W. Rauch和Stuart J. Le Grice）。[对应于2007年4月HIV耐药项目实地考察报告中的Le Grice Project 1]

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, one of four currently undertaken by our section, 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]