Regulation of West Nile Virus Replication

描述(申请人提供)：黄病毒是正链RNA，节肢动物传播的病毒。已知的70种黄病毒中有近一半与人类疾病有关，其中许多病毒经常导致严重的人类发病率和死亡率。圣路易斯脑炎、登革热和西尼罗河病毒(WNV)在美国最近爆发期间感染了人类。目前对黄病毒启动和调节RNA合成或翻译的分子机制知之甚少。预测的保守的病毒RNA序列和结构元件以及与特定细胞蛋白和病毒非结构蛋白的相互作用可能在调节这些过程中发挥重要作用。虽然黄病毒基因组RNA中保守的3‘SL和相邻的CS1序列对病毒复制是必不可少的，但对该区域包含的单个功能元件知之甚少。我们建议通过在西尼罗河病毒感染性克隆中通过突变来测试预测的保守3‘RNA结构和序列中单个NTS的顺式活性，并测试预测的替代RNA三级结构的相关性。还将测试单个NT在细胞蛋白EF-1α的三个精细定位结合部位的顺式活性，这三个结合部位以前被证明与病毒RNA的这一区域相互作用。体外翻译系统和自主的西尼罗河病毒非偶联复制子将分别用于评估单个顺式作用的NT参与病毒RNA翻译和/或复制的调控。拟议的研究将为控制黄病毒RNA翻译和复制的调控机制以及细胞蛋白相互作用在这些过程中的参与提供新的见解。拟议的研究还将确定设计减毒重组候选活疫苗和开发新的抗病毒策略的新靶点。

DESCRIPTION (provided by applicant): Flaviviruses are positive-stranded RNA, arthropod-borne viruses. Nearly half of the 70 known flaviviruses are associated with human disease and many of these regularly cause significant human morbidity and mortality. St. Louis encephalitis, dengue and West Nile virus (WNV), have infected humans during recent outbreaks in the United States. Little is currently known about the molecular mechanisms utilized by flaviviruses to initiate and regulate RNA synthesis or translation. Predicted conserved viral RNA sequence and structure elements and interactions with particular cell proteins as well as viral nonstructural proteins may play essential roles in regulating these processes. Although the conserved 3' SL and adjacent CS1 sequence in the flavivirus genomic RNA have been shown to be essential for virus replication, little is known about the individual functional elements contained in this region. We propose to test the cis-activity of individual nts within predicted conserved 3' RNA structures and sequences by mutation in a WNV infectious clone and to also test the relevance of predicted alternative RNA tertiary structures. The cis-activity of individual nts in three fine mapped binding sites for the cell protein, EF-1alpha, previously shown to interact with this region of the viral RNA will also be tested. An in vitro translation system and an autonomous WNV uncoupled replicon will be used to assess the participation of individual cis-acting nts in the regulation of viral RNA translation and/or replication, respectively. The proposed studies will provide new insights about the regulatory mechanisms that control flavivirus RNA translation and replication as well as about the participation of cell protein interactions in these processes. The proposed studies will also identify novel targets for designing attenuated recombinant candidate live vaccines and for the development of novel antiviral strategies.