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(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‘端含有一个1型帽子(M7GpppAmG)。我们最近发现了一种普遍的机制，即黄病毒NS5甲基化第一个转录的核苷酸上的鸟嘌呤N7和核糖2‘-OH。我们还发现N7甲基转移酶(MTase)是黄病毒复制所必需的。了解CAP甲基化的机制对于开发该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，并开发抗西尼罗病毒的治疗药物。本应用的目的是研究黄病毒MTase的分子机制。我们将以西尼罗河病毒(WNV)为模型来实现三个目标。目标1是定义西尼罗河病毒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顺序进行的，我们假设在这两个顺序的甲基化过程中，底物GpppA-RNA应该被重新定位以接受来自甲基供体S-腺苷-蛋氨酸的N7和2’-O甲基基团。我们将通过确定黄病毒MTase催化顺序反应的原因和方式来检验这一假设。将考察三种模型，包括易位模型、解离-再解离模型和二聚化模型。将定义两个甲基化事件切换过程中的分子细节。目标2和目标3是确定病毒MTase与N7病毒RNA底物(目标2)和2‘-O病毒RNA底物(目标3)形成的复合体的晶体结构。我们最近发现，黄病毒MTase甲基化N7和2‘-O甲基化的病毒RNA序列具有明显的特异性。在目标2和目标3下，我们将在与病毒RNA底物(包括N7和2‘-O底物)和模拟甲基供体分子的复合体中结晶和解决WNV MTase的结构。共晶结构将揭示MTase在N7和2‘-O甲基化过程中如何识别它们的RNA底物。这项研究具有创新性和重要意义，因为在所有已知的MTase中，黄病毒MTase是唯一的，其中一个MTase结构域以病毒RNA依赖的方式催化两个不同的N7和2‘-O甲基化。这里提出的研究有望揭示互补的结果，以确定黄病毒MTase的机制。拟议的工作也很符合R21筹资机制：风险很高，但如果完成，结果会很大。所获得的知识将极大地促进我们对黄病毒帽甲基化的理解，并从长远来看，将为设计黄病毒MTase抑制剂用于抗病毒治疗奠定基础。与公共卫生相关：许多黄病毒会导致重大的人类疾病。其中，登革热、黄热病、西尼罗河、日本脑炎和壁虱传播脑炎被归类为NIAID优先病原体。目前还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)是抗病毒治疗的新靶点。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疗法和抗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