Mechanism of RNA synthesis and 5'-capping by dengue virus NS5 polymerase

登革热病毒 NS5 聚合酶合成 RNA 和 5-加帽的机制

• 批准号: 8240022
• 负责人: Kyung H Choi
• 金额: $ 38.28万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-10 至 2016-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8240022
• 关键词: Antiviral Agents; Antiviral Therapy; Binding; Biochemical; Biochemical Genetics; Bypass; Catalysis; Categories; Collaborations; Communicable Diseases; Complex; Coupled; Coupling; DNA-Directed RNA Polymerase; Data; Dengue; Dengue Virus; Development; Disease; Double-Stranded RNA; Enzymes; Flavivirus; Flavivirus Infections; Foundations; Genome; Goals; Health; Heart; Human; In Vitro; Intervention; Japanese Encephalitis Viruses; Japanese encephalitis virus; Knowledge; Length; Methods; Methylation; Methyltransferase; Modeling; Molecular; Mutate; Mutation; Pathway interactions; Polymerase; Process; Proteins; RNA; RNA Caps; RNA Helicase; RNA chemical synthesis; RNA methylation; RNA replication; RNA-Directed RNA Polymerase; Reaction; Recombinant Proteins; Relative (related person); Replication-Associated Process; Resolution; Resources; Roentgen Rays; Site-Directed Mutagenesis; Solutions; Staging; Structure; Techniques; Testing; Therapeutic; Transcript; Transferase; Vaccines; Viral; Viral Genome; Virus; Virus Replication; West Nile virus; Yellow Fever; base; design; experience; molecular shape; molecular size; multidisciplinary; pathogen; polypeptide; positional cloning; protein structure; public health relevance; replicase; structural biology; therapeutic target

DESCRIPTION (provided by applicant): Flaviviruses including dengue, West Nile, yellow fever, and Japanese encephalitis viruses, pose significant threats as emerging diseases and potential bioterror agents. Despite the considerable impact of flavivirus infection on world-wide health, no antiviral therapies are available, and existing flavivirus vaccines are of limited utility. The long-term goal of this project is to develop antiviral therapeutics based on structural and biochemical information regarding the flavivirus replicase complex. Since replicase complexes are absolutely required for virus replication, knowledge of their structures and mechanisms should identify potential targets for therapeutics and suggest strategies for intervention against threats from these infectious diseases. The flavivirus replicase complex, consisting of a virally-encoded RNA polymerase and helicase as well as other viral and unidentified cellular proteins, is responsible for copying the viral genome. During replication, 5'-RNA capping of the nascent strand occurs along with RNA synthesis. The viral NS5 polymerase has both RNA-dependent RNA polymerase (RdRp) and 5'-RNA methyltransferase (MTase) activities within a single polypeptide, indicating that the RNA synthesis and capping processes may be coupled. Little is known as to whether or how the two activities are coordinated during replication, largely due to a lack of detailed structural information about the full-length polymerase. In Aim 1, the interactions of the RdRp and MTase domains within dengue virus NS5 and the conformational changes that occur therein during the polymerase and methylation reactions, will be determined by small-angle X-ray scattering in solution (Aim 1a). The resulting NS5 model will be tested in recombinant protein and in the infectious virus using site-directed mutagenesis (Aim 1b). In Aim 2, interactions of the full-length NS5 polymerase with RNA will be investigated. A 'dual' substrate that can bind simultaneously to both the RdRp and MTase domains will be designed and tested for its binding to the full- length NS5. In Aim 3, the X-ray crystal structures of the full-length dengue NS5 polymerase and its RNA complexes at different steps along the catalytic pathway will be determined. The combined structural, biochemical, and virological studies will help elucidate the mechanisms for RNA synthesis and its potential coordination with capping reactions in the NS5 polymerase. PUBLIC HEALTH RELEVANCE: Flaviviruses such as dengue virus pose significant threats as emerging diseases and potential bioterror agents. We will perform integrated structural, biochemical, and virological studies on the dengue virus polymerase that carries out viral genome replication processes. The results of our studies will lay the foundation for developing antiviral therapeutics to treat these currently untreatable and often deadly infectious diseases.

描述（由申请人提供）：黄病毒，包括登革热病毒、西尼罗河病毒、黄热病病毒和日本脑炎病毒，作为新出现的疾病和潜在的生物恐怖分子构成了重大威胁。尽管黄病毒感染对全世界健康产生相当大的影响，但尚无可用的抗病毒疗法，并且现有的黄病毒疫苗的效用有限。该项目的长期目标是根据黄病毒复制酶复合物的结构和生化信息开发抗病毒疗法。由于复制酶复合物是病毒复制所必需的，因此对其结构和机制的了解应该能够确定潜在的治疗靶标，并提出针对这些传染病威胁的干预策略。黄病毒复制酶复合物由病毒编码的 RNA 聚合酶和解旋酶以及其他病毒和未鉴定的细胞蛋白组成，负责复制病毒基因组。在复制过程中，新生链的 5'-RNA 加帽与 RNA 合成同时发生。病毒 NS5 聚合酶在单个多肽内同时具有 RNA 依赖性 RNA 聚合酶 (RdRp) 和 5'-RNA 甲基转移酶 (MTase) 活性，表明 RNA 合成和加帽过程可能是耦合的。关于这两种活性在复制过程中是否或如何协调的情况知之甚少，这主要是由于缺乏有关全长聚合酶的详细结构信息。在目标 1 中，登革热病毒 NS5 内 RdRp 和 MTase 结构域的相互作用以及聚合酶和甲基化反应期间发生的构象变化将通过溶液中的小角 X 射线散射来确定（目标 1a）。由此产生的 NS5 模型将使用定点诱变在重组蛋白和感染性病毒中进行测试（目标 1b）。在目标 2 中，将研究全长 NS5 聚合酶与 RNA 的相互作用。将设计可同时结合 RdRp 和 MTase 结构域的“双重”底物，并测试其与全长 NS5 的结合。在目标 3 中，将确定全长登革热 NS5 聚合酶及其 RNA 复合物在催化途径不同步骤的 X 射线晶体结构。结构、生化和病毒学的综合研究将有助于阐明 RNA 合成的机制及其与 NS5 聚合酶中加帽反应的潜在协调。 公共卫生相关性：登革热病毒等黄病毒作为新出现的疾病和潜在的生物恐怖分子构成了重大威胁。我们将对执行病毒基因组复制过程的登革热病毒聚合酶进行综合结构、生化和病毒学研究。我们的研究结果将为开发抗病毒疗法奠定基础，以治疗这些目前无法治疗且往往致命的传染病。