Regulation of West Nile Virus Replication

性状（由申请方提供）：黄病毒属正链RNA，节肢动物传播病毒。在70种已知的黄病毒中，近一半与人类疾病有关，其中许多经常引起显著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。预测保守的病毒RNA序列和结构元件以及与特定细胞蛋白以及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和邻近的CS 1序列已被证明是病毒复制所必需的，但对该区域所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突变来测试预测的保守3' RNA结构和序列内的单个nt的顺式活性，并且还测试预测的替代RNA三级结构的相关性。还将检测细胞蛋白EF-1 α的三个精细定位结合位点中单个nt的顺式活性，先前显示EF-1 α与病毒RNA的该区域相互作用。体外翻译系统和自主WNV解偶联复制子将分别用于评估单个顺式作用nt参与病毒RNA翻译和/或复制的调节。拟议的研究将提供有关控制黄病毒RNA翻译和复制的调控机制以及细胞蛋白相互作用参与这些过程的新见解。拟议的研究还将确定用于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.