Characterization of hantavirus N protein-mediated translation mechanism

描述(申请人提供)：汉坦病毒，布尼亚病毒科的成员是被包膜的负链新出现的RNA病毒和A类病原体，通过受感染啮齿动物的雾化排泄物传播给人类。汉坦病毒已经进化出一种独特的翻译机制，由病毒核衣壳蛋白(N)操纵，这种机制优先促进病毒mRNAs在宿主细胞中的翻译，在宿主细胞中，细胞转录产物竞争相同的翻译机制。这种翻译机制在概念上不同于迄今已知的所有翻译机制，并且有助于在不需要eIF4F帽结合复合体的情况下翻译带帽的mRNAs。汉坦病毒mRNAs含有一个独特的42-52核苷酸长的5‘非翻译区，在5’端含有一个高度保守的三联体重复序列，与三聚体N蛋白特异结合。此外，N还通过位于40S亚基头部的核糖体蛋白S19(RPS19)与40S核糖体亚基结合。N优先将40S核糖体亚基装载到病毒mRNA5‘端非编码区，并促进其体外翻译。我们将进一步研究5‘非编码区在N介导的细胞内翻译机制中的作用。我们将确定汉坦病毒mRNAs的翻译是否特定地依赖于N介导的翻译机制。我们还将识别和表征N蛋白中的RPS19结合域，并证明N-RPS19相互作用在N介导的翻译策略中的作用。我们将确定N蛋白是否仍然与多聚体相关的mRNAs中80S核糖体的40S亚基相关。这些研究将揭示一种新的翻译启动机制，可能大多数负链RNA病毒在感染过程中利用这种机制来促进其mRNAs在宿主细胞中的翻译。拟议的研究还将揭示未来抗病毒药物设计的新靶点。 公共卫生相关性：目前尚无汉坦病毒相关疾病的治疗方法。这项应用的主要目标是描绘汉坦病毒在细胞内复制的机制。此外，拟议的研究将有助于确定抗病毒药物设计的目标。

DESCRIPTION (provided by applicant): Hantaviruses, members of the Bunyaviridae family are enveloped negative stranded emerging RNA viruses and category A pathogens that are transmitted to humans through aerosolized excreta of infected rodents. Hantaviruses have evolved a unique translation mechanism, operated by viral nucleocapsid protein (N), which preferentially facilitates the translation of viral mRNAs in host cells where cellular transcripts are competing for the same translation machinery. This translation mechanism is conceptually different from all translation mechanisms known to date, and facilitates the translation of capped mRNAs without the requirement of eIF4F cap binding complex. Hantaviral mRNAs contain a unique 42-52 nucleotide long 5' untranslated region (UTR) harboring a highly conserved triplet repeat sequence at the 5' terminus that specifically binds to the trimeric N protein. In addition, N also binds to the 40S ribosomal subunit via the ribosomal protein S19 (RPS19), located at the head region of 40S subunit. N preferentially loads 40S ribosomal subunits onto the viral mRNA 5' UTR and facilitates their translation in vitro. We will further characterize the role of 5' UTR in the translation of viral mRNAs by N-mediated translation mechanism in cells. We will determine whether hantaviral mRNAs specifically depend on N-mediated translation mechanism for their translation. We will also identify and characterize the RPS19 binding domain in N protein and demonstrate the role of N-RPS19 interaction in N-mediated translation strategy. We will determine whether N protein remains associated with the 40S subunit of 80S ribosome in polysome associated mRNAs. These studies will shed light on a novel mechanism of translation initiation that likely most negative stranded RNA viruses use to favor the translation of their mRNAs in host cells during infection. The proposed studies will also reveal novel targets for antiviral drug design in future. PUBLIC HEALTH RELEVANCE: There is no treatment for hantavirus associated disease at present. The major goal of this application is to delineate the mechanism of hantavirus replication in cells. In addition, the proposed studies will help in the identification of targets for antiviral drug design.