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

描述（由申请人提供）：涉及夺帽的病毒转录起始的范例基于正粘病毒、流感病毒，并假定异源三聚体病毒RNA依赖性RNA聚合酶（RdRp）通过流感病毒RdRp的核酸内切酶活性从细胞mRNA获得5'帽。所有负链节段RNA病毒（包括布尼亚病毒和沙粒病毒）都假定了这种一般的夺帽机制，尽管它们的RdRps结构不同，并且它们在宿主细胞内的不同位置复制。由于病毒RdRp在转录起始期间使用加帽的寡核糖核苷酸作为引物，因此病毒必须有效地与细胞mRNA去帽机制竞争以保留这些mRNA帽以用于病毒mRNA的有效合成。我们的初步研究表明，汉坦病毒核衣壳蛋白（N）与细胞mRNA的5'端帽结合，并保护其从5'端降解。长度至少为180个核苷酸的受保护的5'加帽寡核苷酸通过N.这些封闭在P体中的加帽寡核苷酸有效地用作复制Sin Nombre汉他病毒的引物，用于合成病毒mRNA。由于病毒RdRp在转录起始期间使用的RNA引物的长度在6-18个核苷酸之间变化，因此必须对P体中的隔离的加帽寡核苷酸进行进一步修剪以产生适当长度和特异性的加帽RNA引物。我们假设这种修剪所需的核酸内切酶活性存在于病毒RdRp中。由于N以高亲和力结合mRNA帽，因此N可能与病毒RdRp结合产生RNA引物。我们还假设，隔离加帽寡核苷酸在P体的基础上选择的5'帽附近的核苷酸序列作为引物的病毒mRNA合成的起始。汉坦病毒复制极有可能也发生在P体中。多方面的实验方法已被设计来检查这些假设。我们有强有力的初步数据表明，N具有不同的帽结合和RNA结合结构域。因此，需要鉴定和表征N的帽结合结构域。这些研究将有助于理解布尼亚病毒和沙粒病毒致病的分子机制，重点是转录起始的夺帽机制。在未来，这些研究还将有助于鉴定抗病毒治疗的靶点，并设计未来潜在的抗病毒药物，用于由负链RNA病毒引起的疾病，负链RNA病毒使用夺帽机制启动病毒mRNA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“抢帽”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以及设计未来治疗HPS的潜在抗病毒药物。

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.