The West Nile virus methyltransferase

西尼罗河病毒甲基转移酶

• 批准号: 7842651
• 负责人: HONGMIN LI
• 金额: $ 18.56万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842651
• 关键词: Antiviral Agents; Antiviral Therapy; Binding; Binding Sites; Biochemical Genetics; Complex; Crystallization; Dengue; Development; Dimerization; Dissociation; Ensure; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Epidemic; Event; Flavivirus; Flavivirus Infections; Fluorescence Resonance Energy Transfer; Funding Mechanisms; Genome; Goals; Guanine; Human; Intervention; Japanese Encephalitis; Knowledge; Mediating; Methionine; Methylation; Methyltransferase; Modeling; Molecular; Molecular Mechanisms of Action; Mutagenesis; National Institute of Allergy and Infectious Disease; Nature; Nucleotides; Outcome; Positioning Attribute; RNA; RNA Caps; RNA Sequences; Reaction; Research; Research Personnel; Ribose; Specificity; Structure; Surface; System; Testing; Therapeutic; Therapeutic Intervention; Tick-Borne Encephalitis; Tick-Borne Encephalitis Virus; Tick-Borne Encephalitis Viruses; Time; United States; Vaccine Therapy; Virus Replication; West Nile virus; Work; Yellow Fever; analog; analytical ultracentrifugation; design; enzyme substrate complex; genetic analysis; high risk; human disease; inhibitor/antagonist; innovation; member; methyl group; mutant; novel; pathogen; public health relevance; three dimensional structure; viral RNA

DESCRIPTION (provided by applicant): Many members of the genus Flavivirus are significant human pathogens. The 5' end of flavivirus plus- sense RNA genome contains a type 1 cap (m7GpppAmG). We recently found a general mechanism that the flavivirus NS5 methylates the guanine N7 on the cap and ribose 2'-OH on the first transcribed nucleotide. We also found that the N7 methyltransferase (MTase) is essential for flavivirus replication. Understanding the mechanism of cap methylation is critical for development of inhibitors of this enzyme for antiviral intervention. Our long-range goal is to study the molecular mechanisms of flaviviral replication and to develop anti-WNV therapeutics. The objective of this application is to study the molecular mechanism of flavivirus MTase. We will use West Nile Virus (WNV) as a model to accomplish three aims. Aim 1 is to define the "repositioning" mechanism of the WNV RNA cap methylation. Because of the nature that the two N7 and 2'-O methylations are sequential by one MTase, we hypothesize that substrate GpppA-RNA should be "repositioned" to accept the N7 and 2'-O methyl groups from the methyl donor, S-Adenosyl-Methionine (SAM), during the two sequential methylations. We will test the hypothesis by determining why and how the flavivirus MTases catalyze the sequential reactions. Three models including a translocation model, a dissociation-and-reassociation model, and a dimerization model will be examined. The molecular details during the switch of the two methylation events will be defined. Aims 2 and 3 are to determine crystal structures of favivirus MTase in complex with an N7 viral RNA substrate (Aim 2) and with a 2'-O viral RNA substrate (Aim 3). We recently found that flavivirus MTase methylates cap structures with distinct specificities for viral RNA sequences for the N7 and 2'-O methylations, respectively. Under Aims 2 and 3, we will crystallize and solve the structures of the WNV MTase in complex with viral RNA substrates (including both N7 and 2'-O substrates) and analog methyl donor molecule. The co-crystal structure will reveal how the MTase recognizes their RNA substrates during N7 and 2'-O methylations. The proposed research is innovative and significant, because the flavivirus MTase is unique among all known MTases in which one MTase domain catalyzes two distinct N7 and 2'-O methylations in a viral RNA-dependent manner. The studies proposed here are expected to reveal complementary results to define the mechanism of the flavivirus MTase. The proposed work also fits well with the R21 funding mechanism: high risk but with significant outcome if accomplished. The knowledge acquired will significantly advance our understanding of flavivirus cap methylation, and in the long term, will lay the groundwork for designing inhibitors of the flavivirus MTase for antiviral therapy. PUBLIC HEALTH RELEVANCE: Many flaviviruses cause significant human diseases. Among then, dengue, yellow fever, West Nile, Japanese encephalitis, and tick-borne encephalitis are categorized as the NIAID Priority Pathogens. No effective antiviral therapy is currently available for treatment of flavivirus infections. We recently found that flavivirus methyltransferase (MTase) is a novel target for antiviral therapy. The current proposal is to define the molecular mechanism and function of the flavivirus MTase. These studies are expected to provide critical information for development of MTase therapies and vaccines against flaviviruses.

描述（由申请人提供）：黄病毒属的许多成员是重要的人类病原体。黄病毒正义RNA基因组的5'端含有1型帽（m7 GpppAmG）。我们最近发现了黄病毒NS 5甲基化帽上的鸟嘌呤N7和第一个转录核苷酸上的核糖2 '-OH的一般机制。我们还发现，N7甲基转移酶（MTase）是黄病毒复制所必需的。了解帽甲基化的机制对于开发这种酶的抑制剂用于抗病毒干预至关重要。我们的长期目标是研究黄病毒复制的分子机制，并开发抗西尼罗河病毒的治疗方法。本研究的目的是研究黄病毒MTase的分子机制。我们将使用西尼罗河病毒（WNV）作为一个模型，以实现三个目标。目的1是明确西尼罗河病毒RNA帽甲基化的“重新定位”机制。由于两个N7和2 '-O甲基化是由一个MTase连续的性质，我们假设在两个连续的甲基化过程中，底物GpppA-RNA应该被“重新定位”以接受来自甲基供体S-腺苷-甲硫氨酸（SAM）的N7和2'-O甲基。我们将通过确定黄病毒MT酶催化顺序反应的原因和方式来检验这一假设。三个模型，包括易位模型，解离和再结合模型，和二聚体模型将被检查。将定义两个甲基化事件转换期间的分子细节。目的2和3是确定与N7病毒RNA底物（目的2）和与2 '-O病毒RNA底物（目的3）复合的黄病毒MTase的晶体结构。我们最近发现，黄病毒MTase甲基化帽结构，分别对N7和2 '-O甲基化的病毒RNA序列具有不同的特异性。在目标2和3下，我们将结晶并解析与病毒RNA底物（包括N7和2 '-O底物）和类似物甲基供体分子复合的WNV MTase的结构。共晶体结构将揭示MTase如何在N7和2 '-O甲基化期间识别其RNA底物。这项研究具有创新性和重要意义，因为黄病毒MTase在所有已知的MTase中是独一无二的，其中一个MTase结构域以病毒RNA依赖的方式催化两种不同的N7和2 '-O甲基化。这里提出的研究预计将揭示互补的结果，以确定黄病毒MTase的机制。拟议的工作也非常符合R21供资机制：风险高，但如果完成，将取得重大成果。所获得的知识将大大提高我们对黄病毒帽甲基化的理解，从长远来看，将为设计黄病毒MTase抑制剂用于抗病毒治疗奠定基础。公共卫生相关性：许多黄病毒引起重大人类疾病。其中，登革热、黄热病、西尼罗河病毒、日本脑炎和蜱传脑炎被归类为NIAID优先病原体。目前没有有效的抗病毒疗法可用于治疗黄病毒感染。我们最近发现，黄病毒甲基转移酶（MTase）是一个新的抗病毒治疗的目标。目前的建议是确定黄病毒MTase的分子机制和功能。这些研究有望为MTase治疗和黄病毒疫苗的开发提供关键信息。

项目成果

Flavivirus RNA cap methyltransferase: structure, function, and inhibition.

• DOI: 10.1007/s11515-010-0660-y
• 发表时间: 2010-08-01
• 期刊: Frontiers in biology

Inexpensive electronics and software for photon statistics and correlation spectroscopy.

• DOI: 10.1119/1.4869188
• 发表时间: 2014-07
• 期刊: American journal of physics
• 影响因子: 0.9
• 作者: Gamari BD;Zhang D;Buckman RE;Milas P;Denker JS;Chen H;Li H;Goldner LS
• 通讯作者: Goldner LS

Selective inhibition of the West Nile virus methyltransferase by nucleoside analogs.

• DOI: 10.1016/j.antiviral.2012.12.012
• 发表时间: 2013-03
• 期刊: ANTIVIRAL RESEARCH
• 影响因子: 7.6
• 作者: Chen, Hui;Liu, Lihui;Jones, Susan A.;Banavali, Nilesh;Kass, Jorden;Li, Zhong;Zhang, Jing;Kramer, Laura D.;Ghosh, Arun K.;Li, Hongmin
• 通讯作者: Li, Hongmin