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

描述（由申请人提供）：病毒转录起始涉及帽撷取的范例是基于正粘病毒，流感病毒，并假设异三聚体病毒RNA依赖RNA聚合酶（RdRp）通过流感RdRp的内切酶活性从细胞mrna中获得5'帽。尽管所有负链分段RNA病毒（包括布尼亚病毒和沙粒病毒）的RdRps在结构上不同，并且在宿主细胞内的不同位置复制，但人们认为这种一般的cap抢夺机制适用于所有负链分段RNA病毒。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，因此病毒必须有效地与细胞mRNA脱帽机制竞争，以保存这些mRNA帽，以便有效地合成病毒mRNA。我们的初步研究表明，汉坦病毒核衣壳蛋白(N)以高亲和力与细胞mrna的5′端结合，并保护其从5′端降解。n将至少180个核苷酸长度的受保护的5'带帽寡核苷酸储存在细胞P小体中，这些被隔离在P小体中的带帽寡核苷酸被有效地用作引物，用于复制Sin Nombre汉坦病毒以合成病毒mRNA。由于病毒RdRp在转录起始过程中使用的RNA引物长度从6-18个核苷酸不等，因此必须对P小体中隔离的带帽寡核苷酸进行进一步修剪，以产生适当长度和特异性的带帽RNA引物。我们假设这种修剪所需的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一起产生RNA引物。我们还假设，P小体中隔离的带帽寡核苷酸是根据5'帽附近的核苷酸序列选择的，作为启动病毒mRNA合成的引物。汉坦病毒很可能也在P体中进行复制。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具有不同的cap结合域和RNA结合域。因此，鉴定和表征N的帽结合域是必要的。这些研究将有助于了解布尼亚病毒和沙粒病毒发病的分子机制，重点研究转录起始的抓帽机制。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，用于使用夺帽机制启动病毒mRNA合成的负链RNA病毒引起的疾病。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）死亡率为50%。“cap snatching”的研究将有助于了解汉坦病毒的发病机制，确定抗病毒治疗的靶点，以及设计治疗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.