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（由申请人提供）：涉及盖帽的病毒转录启动的范例基于原肌霉菌病毒，流感，并认为异核三聚体病毒RNA依赖性RNA RNA聚合酶（RDRP）通过甲型核酸内核酸内核酸杆菌活性的细胞mRNA获得5'的CAPS。尽管它们的RDRP在结构上不同，并且它们在宿主细胞内的不同位置复制，但已假定所有负滞留的RNA病毒（包括Bunyaviruses和arthaviruses）在内的所有负链分割的RNA病毒中都假定了这种通用的盖抓扣机制。由于病毒RDRP在转录启动过程中使用限制的寡核苷酸作为底漆，因此病毒必须有效地与细胞mRNA脱粉机器竞争，以保留这些mRNA帽，以有效地合成病毒mRNA。我们的初步研究表明，汉塔病毒核素蛋白（N）与细胞mRNA的5'帽高亲和力结合，并保护其从5'末端的降解。在长度上，至少180个核苷酸的受保护的5'上限寡聚物被N存储在细胞体中。通过复制sin nombre hantavirus以合成病毒mRNA的合成，这些封闭的寡聚物被隔离在P体中。由于转录起始过程中病毒RDRP使用的RNA引物的长度因6-18个核苷酸而异，因此必须在P体中进一步修剪隔离的限制寡核体，以生成具有适当长度和特异性的盖帽RNA引物。我们假设这种修剪需要的核酸内切酶活性位于病毒RDRP中。由于n结合具有高亲和力的mRNA盖，因此n可能与病毒RDRP结合起作用，以产生RNA底漆。我们还假设，在5'CAP附近选择了P体中隔离的限制的寡寡寡做，以作为启动病毒mRNA合成的引物。汉坦病毒复制也很有可能发生在P体中。多方面的实验方法已设计用于检查这些假设。我们有强大的初步数据，表明N具有独特的CAP结合和RNA结合域。因此，有必要识别和表征n的盖结合域。这些研究将有助于理解Bunya和Arravirus发病机理的分子机制，重点是转录启动的盖机理。将来，这些研究还将有助于鉴定抗病毒疗法的靶标，并设计由负链RNA病毒引起的未来潜在抗病毒药物，这些疾病使用使用CAP抢断机制来启动病毒mRNA合成。公共卫生相关性：由啮齿动物传播类别引起的汉塔病毒心脏肺综合症（HPS）病毒死亡率为50％。有关“盖帽抢夺”的研究将有助于理解汉塔病毒发病机理的机制，鉴定抗病毒治疗的靶标以及未来潜在抗病毒药物治疗HPS的靶标。