Role of Cellular P-bodies and Hantavirus Nucleocapsid Protein in Viral mRNA "cap

细胞P体和汉坦病毒核衣壳蛋白在病毒mRNA“帽”中的作用

• 批准号: 7897814
• 负责人: Mohammad A Mir
• 金额: $ 18.75万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-22 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7897814
• 关键词: Affinity; Antiviral Agents; Antiviral Therapy; Arenavirus; Base Sequence; Binding; Binding Sites; Categories; Cell Nucleus; Cell physiology; Cells; Characteristics; Cleaved cell; Data; Disease; Exonuclease; Future; Generations; Genes; Genetic Transcription; Genome; Goals; Hantavirus; Hantavirus Pulmonary Syndrome; Influenza; Length; Location; Lung; Maps; Messenger RNA; Modeling; Molecular; N Domain; Nucleocapsid Proteins; Nucleotides; Oligoribonucleotides; Orthobunyavirus; Orthomyxoviridae; Pathogenesis; Process; Qualifying; RNA Binding; RNA Caps; RNA Recognition Motif; RNA Viruses; RNA primers; RNA-Directed RNA Polymerase; Rodent; Role; Sin Nombre virus; Specificity; Syndrome; Testing; Therapeutic; Transcription Initiation; Viral; Viral Genome; Virus; Work; base; decapping enzyme; design; endonuclease; flu activity; influenzavirus; mRNA Transcript Degradation; mRNA capping; mRNA decapping; mortality; prevent; public health relevance; therapy design; viral RNA

DESCRIPTION (provided by applicant): The paradigm for viral transcription initiation involving cap snatching is based on orthomyxovirus, influenza, and posits that heterotrimeric viral RNA dependent RNA polymerase (RdRp) acquires 5' caps from cellular mRNAs through the endonuclease activity of influenza RdRp. This general cap snatching mechanism has been assumed for all minus stranded segmented RNA viruses including the bunyaviruses and arenaviruses, although their RdRps are structurally different and they replicate at different locations inside the host cell. Since viral RdRp uses capped oligoribonucleotides as primers during transcription initiation, viruses have to effectively compete with the cellular mRNA decapping machinery to preserve these mRNA caps for the efficient synthesis of viral mRNAs. Our preliminary studies show that hantavirus nucleocapsid protein (N) binds with high affinity to the 5' caps of cellular mRNAs and protects their degradation from 5' termini. The protected 5' capped oligos of at least 180 nucleotides in length are stored in cellular P bodies by N. These capped oligos sequestered in P bodies are efficiently used as primers by replicating Sin Nombre hantavirus for the synthesis of viral mRNA. Since the length of RNA primers used by viral RdRp during transcription initiation varies from 6-18 nucleotides, further trimming of sequestered capped oligos in P bodies must take place for the generation of capped RNA primer of appropriate length and specificity. We hypothesize that endonuclease activity required for such trimming resides in viral RdRp. Since N binds the mRNA caps with high affinity, it is possible that N works in conjunction with viral RdRp in generating the RNA primer. We also hypothesize that sequestered capped oligos in P bodies are selected on the basis of nucleotide sequence in the vicinity of 5' cap to serve as primers for the initiation of viral mRNA synthesis. It is highly likely that hantavirus replication also takes place in P bodies. Multifaceted experimental approaches have been designed to check these hypotheses. We have strong preliminary data showing that N has distinct cap binding and RNA binding domains. Hence identification and characterization of cap binding domain of N is warranted. These studies will help in understanding the molecular mechanism of bunya and arenavirus pathogenesis with a focus on cap snatching mechanism of transcription initiation. In future these studies will also help in the identification of targets for the antiviral therapeutics and design of future potential antiviral agents for diseases caused by negative stranded RNA viruses which use cap snatching mechanism for the initiation of viral mRNA synthesis. PUBLIC HEALTH RELEVANCE: Hantavirus cardio pulmonary syndrome (HPS) caused by rodent borne category A virus has a mortality of fifty percent. The studies on "cap snatching" will help in understanding the mechanism of hantavirus pathogenesis, identification of targets for the antiviral therapy and design of future potential antiviral agents for the treatments of HPS.

描述(申请人提供)：涉及帽子抢夺的病毒转录启动范例基于正粘病毒、流感，并假设异源三聚体病毒RNA依赖RNA聚合酶(RdRp)通过流感RdRp的内切酶活性从细胞mRNAs获得5‘CAP。这一一般的帽子抢夺机制已被假设为所有负链节段RNA病毒，包括布尼亚病毒和阿雷纳病毒，尽管它们的RdRp结构不同，它们在宿主细胞内的不同位置复制。由于病毒RdRp在转录起始过程中使用带帽的寡核苷酸作为引物，病毒必须有效地与细胞内的mRNA解译机制竞争，以保存这些mRNA的帽子，从而有效地合成病毒的mRNAs。我们的初步研究表明，汉坦病毒核衣壳蛋白(N)与细胞mRNAs的5‘端高亲和力结合，并保护其从5’端开始降解。受保护的至少180个核苷酸的5‘端封帽寡核苷酸被N储存在细胞内的P小体中，这些封顶寡核苷酸被有效地用作复制新城疫汉坦病毒以合成病毒mRNA的引物。由于病毒RdRp在转录起始过程中使用的RNA引物的长度从6-18个核苷酸不等，因此必须进一步修剪P小体中隔离的封端寡核苷酸，才能产生合适长度和特异性的封端RNA引物。我们假设这种修剪所需的内切酶活性驻留在病毒RdRp中。由于N以高亲和力结合mRNA帽，因此N有可能与病毒RdRp一起产生RNA引物。我们还假设，P小体中隔离的封端寡核苷酸是根据5‘端附近的核苷酸序列选择的，作为启动病毒mRNA合成的引物。汉坦病毒复制很可能也发生在P小体中。已经设计了多方面的实验方法来检验这些假设。我们有强有力的初步数据表明，N有不同的帽子结合结构域和RNA结合结构域。因此，鉴定和表征N的帽结合结构域是必要的。这些研究将有助于了解兔细小病毒致病的分子机制，重点是转录起始的帽子抢夺机制。在未来，这些研究还将有助于确定抗病毒治疗的靶点，并设计未来潜在的抗病毒药物，用于治疗由负链RNA病毒引起的疾病，负链RNA病毒利用帽子抢夺机制启动病毒mRNA的合成。公共卫生相关性：由啮齿动物传播的A类病毒引起的汉坦病毒心肺综合征(HPS)的死亡率为50%。对“帽子抢夺”的研究将有助于了解汉坦病毒的致病机制，确定抗病毒治疗的靶点，并设计未来治疗HPS的潜在抗病毒药物。