Viral and Microbial RNA Modifying Enzymes

病毒和微生物 RNA 修饰酶

• 批准号: 8236142
• 负责人: Stewart H Shuman
• 金额: $ 53.9万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8236142
• 关键词: ATP-Binding Cassette Transporters; Active Sites; Amoeba genus; Anti-Infective Agents; Antiviral Agents; Bacteria; Bacterial RNA; Bacteriophage T4; Bacteriophages; Binding; Binding Proteins; Biochemical Reaction; Biological Models; C-terminal; Catalytic Domain; Cells; Cellular Stress; Clostridium thermocellum; Coupled; DNA Viruses; Diphosphates; Enzymes; Escherichia coli; Event; Evolution; Family; Funding; Genetic; Genetic Models; Genetic Translation; Genomics; Glutamic Acid-Specific tRNA; Grant; Guanine; Healed; Homologous Gene; Human; Hydrolysis; Immune response; Infection; Ligase; Light; Lysine-Specific tRNA; Mammalian Cell; Manganese; Maps; Mediating; Messenger RNA; Methyltransferase; Modification; N-terminal; Open Reading Frames; Operon; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphotransferases; Phylogenetic Analysis; Phylogeny; Polynucleotide 5' -Hydroxyl-Kinase; Poxviridae; Property; Proteins; Protozoa; RNA; RNA Caps; RNA Ligase (ATP); RNA Processing; RNA triphosphatase; Reaction; Research; Ribose; Saccharomycetales; Smallpox; Specificity; Structure; Substrate Specificity; System; Taxon; Toxin; Transfer RNA; Vaccinia; Vaccinia virus; Vaccinium; Viral; Viral Proteins; Virus; Virus Diseases; Work; Yeasts; anticodon nuclease; cytotoxicity; drug discovery; fungus; guanylyltransferase; healing; in vivo; insight; killings; mRNA Precursor; mRNA Stability; mRNA capping; mRNA guanylyltransferase; methyl group; microbial; novel; nuclease; nucleoside triphosphatase; pathogen; plant fungi; polypeptide; programs; public health priorities; repair enzyme; repaired; response; restriction enzyme; seal; tripolyphosphate; weapons

DESCRIPTION (provided by applicant): PROJECT SUMMARY: This project aims to illuminate the mechanisms and structures of viral enzymes that cap the 5' end of messenger RNA. The m7GpppN cap is a distinctive feature of eukaryal cellular and viral mRNA that is required for mRNA stability and translation. Capping entails three enzymatic reactions: (i) the 5' triphosphate of the pre-mRNA is hydrolyzed to a diphosphate by RNA triphosphatase (TPase); (ii) the diphosphate RNA end is capped with GMP by RNA guanylyltransferase (GTase); and (iii) the GpppN cap is methylated by RNA (guanine-N7) methyltransferase (MTase). Whereas the three capping reactions are universal in eukarya and DNA viruses, there is a remarkable diversity in the genetic organization of the cap- forming enzymes in different taxa and in different viruses, as well as a complete divergence in the structure and catalytic mechanism of the TPase enzymes found in fungi, protozoa, and several large DNA viruses versus the human TPase. These differences can be exploited to develop novel anti-infective agents directed against capping of the pathogen's mRNAs. They also provide instructive clues to eukaryal phylogeny and evolution of large DNA viruses. This proposal focuses on the poxvirus mRNA capping enzyme, a heterodimer composed of TPase, GTase and MTase domains fused within a single large polypeptide subunit, plus a smaller subunit that binds and stimulates the MTase domain. We plan to functionally map the active sites and domain interfaces of the poxvirus capping enzyme, guided by a new crystal structure of the complete capping enzyme heterodimer. As an outgrowth of our studies of RNA end-modification by viral enzymes, we've developed a new line of research into end-healing and end-sealing enzymes that repair programmed RNA breaks. Programmed RNA damage is an ancient mechanism of responding to cellular stress and distinguishing self from non-self. RNA damage also figures prominently in host responses to virus infection. tRNA damage inflicted by a latent anticodon nuclease PrrC (which breaks tRNALys) underlies a potent host innate immune response to bacteriophage T4 infection, which is thwarted by a virus-encoded tRNA repair system consisting of T4 Pnkp (polynucleotide kinase/phosphatase) and T4 Rnl1 (RNA ligase 1). We have dissected the mechanism, structure, and specificity of Pnkp and Rnl1, and we have extended our analysis to discover or characterize new RNA repair enzymes from viruses, bacteria and human cells. RNA damage and repair now comprise a significant component of the research effort supported by this grant. The current plan focuses on two aspects of the host-pathogen RNA damage/repair dynamic: (i) genetic and structural analysis of PrrC anticodon nucleases and (ii) characterization of a newly discovered two-component RNA repair system (Hen1-Pnkp) that is distributed widely among bacteria. PUBLIC HEALTH RELEVANCE: RELEVANCE: Viruses must solve (or circumvent) the mRNA capping problem in order to produce viral proteins and attain a successful virus-host dynamic in the face of cellular surveillance systems that sense uncapped RNA ends. Poxviruses are cytoplasmic DNA viruses that encode their own mRNA capping machinery. Structural and mechanistic differences between the poxvirus and mammalian capping systems recommend capping as a target for antipoxviral drug discovery. Antipoxviral strategies are a national public health priority in light of concern that undeclared stocks of smallpox could be used as a bioterror weapon.

描述（由申请人提供）： 项目摘要：该项目旨在阐明给信使 RNA 5' 端加帽的病毒酶的机制和结构。 m7GpppN 帽是真核细胞和病毒 mRNA 的一个显着特征，是 mRNA 稳定性和翻译所必需的。加帽需要三个酶促反应：(i) RNA 三磷酸酶 (TPase) 将前 mRNA 的 5' 三磷酸水解为二磷酸； (ii) 二磷酸 RNA 末端通过 RNA 鸟苷基转移酶 (GTase) 用 GMP 加帽； (iii) GpppN 帽被 RNA (鸟嘌呤-N7) 甲基转移酶 (MTase) 甲基化。虽然这三种加帽反应在真核生物和 DNA 病毒中是普遍存在的，但不同类群和不同病毒中加帽酶的遗传组织存在显着的多样性，并且加帽反应的结构和催化机制也完全不同。真菌、原生动物和几种大型 DNA 病毒中发现的 TPase 与人类 TPase 的比较。这些差异可用于开发针对病原体 mRNA 封端的新型抗感染剂。它们还为真核系统发育和大型 DNA 病毒的进化提供了指导性线索。该提案重点关注痘病毒 mRNA 加帽酶，这是一种异二聚体，由融合在单个大多肽亚基内的 TPase、GTase 和 MTase 结构域以及结合并刺激 MTase 结构域的较小亚基组成。我们计划在完整加帽酶异二聚体的新晶体结构的指导下，对痘病毒加帽酶的活性位点和结构域界面进行功能图谱。 作为我们通过病毒酶进行 RNA 末端修饰研究的成果，我们开发了一系列新的研究方向，研究修复程序性 RNA 断裂的末端修复和末端密封酶。程序性 RNA 损伤是一种古老的机制，用于响应细胞压力并区分自我和非自我。 RNA 损伤在宿主对病毒感染的反应中也占有重要地位。潜伏反密码子核酸酶 PrrC（破坏 tRNALys）造成的 tRNA 损伤是宿主对噬菌体 T4 感染产生强大先天免疫反应的基础，而由 T4 Pnkp（多核苷酸激酶/磷酸酶）和 T4 Rnl1 组成的病毒编码的 tRNA 修复系统会阻碍这种免疫反应。 （RNA 连接酶 1）。我们剖析了 Pnkp 和 Rnl1 的机制、结构和特异性，并将我们的分析扩展到发现或表征来自病毒、细菌和人类细胞的新 RNA 修复酶。 RNA 损伤和修复现在是这笔资金支持的研究工作的重要组成部分。目前的计划重点关注宿主-病原体 RNA 损伤/修复动态的两个方面：(i) PrrC 反密码子核酸酶的遗传和结构分析，以及 (ii) 新发现的双组分 RNA 修复系统 (Hen1-Pnkp) 的表征，该系统在细菌中分布广泛。 公共卫生相关性： 相关性：病毒必须解决（或规避）mRNA 加帽问题，才能产生病毒蛋白，并在面对感知未加帽 RNA 末端的细胞监视系统时获得成功的病毒宿主动态。痘病毒是细胞质 DNA 病毒，编码自己的 mRNA 加帽机制。痘病毒和哺乳动物加帽系统之间的结构和机制差异建议将加帽作为抗痘病毒药物发现的目标。鉴于人们担心未申报的天花库存可能被用作生物恐怖武器，抗痘病毒策略是国家公共卫生的优先事项。