Alternative regulation of ISGs in WNV-infected cells

描述（由申请人提供）：许多黄病毒，包括西尼罗病毒（WNV），日本脑炎病毒，蜱传脑炎病毒和登革热病毒，是重要的人类病原体。目前还没有有效的抗病毒治疗方法来治疗黄病毒感染，黄病毒诱导的发病机制也不完全清楚。已经发现了干扰素刺激基因（ISG）亚群存在两种1型IFN和irf -3/7独立的细胞“备份”上调途径的证据，以及一种新的病毒介导的对抗机制，该机制旨在抑制抗病毒ISG亚群的蛋白质产生。建议进行初步探索性研究以确定所涉及的机制。具体目标集中在获得对以下方面的初步理解：(1)在wnv感染细胞中，在缺乏典型I型干扰素信号通路的情况下，irf -3/7独立的isg亚群在感染后12小时上调的机制，以及在wnv感染细胞中显示延迟激活的isg亚群所需的不同调节机制；(2)在西尼罗河病毒感染的细胞中，ISGs第二亚群的蛋白表达受到抑制的机制。在Aim 1中，我们将在WNV感染细胞中快速上调和延迟上调的模型isg启动子中绘制参与干扰素不依赖性isg上调的关键转录因子结合位点（TFBSs）。绘制的TFBSs的功能相关性将通过诱变和体外和体内dna -蛋白质相互作用试验进行测试。结合到所绘制的TFBSs的转录因子将通过MassSpec对DNA下拉获得的蛋白质进行测序来鉴定。在Aim 2下，将研究ISG mRNA稳定性、细胞RNA结合蛋白和细胞mirna在降低西尼罗河病毒感染细胞中ISG亚群表达的蛋白水平中的作用。对这些机制的详细了解将增加对宿主对病毒感染的先天反应的复杂性和一种新的病毒对抗机制的认识。病毒对抗机制的描述将为开发可能适用的抗病毒治疗药物提供新的细胞靶点

DESCRIPTION (provided by applicant): Many flaviviruses, including West Nile virus (WNV), Japanese encephalitis virus, tick- borne encephalitis virus and dengue virus, are significant human pathogens. No effective antiviral therapies currently exist for treating individuals with flavivirus infections and flavivirus-induced pathogenesis is not completely understood. Evidence for the existence of two Type 1 IFN- and IRF-3/7-independent cellular "backup" up-regulation pathways for subsets of interferon stimulated genes (ISG) and for a novel virus- mediated counteraction mechanism directed at suppressing protein production by a subset of antiviral ISGs have been discovered. Initial exploratory studies are proposed to define the mechanisms involved. The specific aims are focused on gaining an initial understanding of: (1) the mechanisms responsible for the up-regulation of a subset of IRF-3/7-independent ISGs by 12 hr after infection in the absence of a canonical Type I interferon signaling pathway in WNV-infected cells and for the different regulatory mechanism required for a second subset of ISGs that show delayed activation in WNV-infected cells; and (2) the mechanism by which protein expression by the second subset of ISGs is suppressed in WNV-infected cells. Under Aim 1, we will map the critical transcription factor binding sites (TFBSs) involved in interferon-independent up-regulation of ISGs in the promoters of model ISGs that are rapidly up-regulated and those that show delayed up-regulation in WNV- infected cells. The functional relevance of the mapped TFBSs will be tested by mutagenesis and by in vitro and in vivo DNA-protein interaction assays. Transcription factors binding to the mapped TFBSs will be identified by MassSpec sequencing of proteins obtained by DNA pull-down. Under Aim 2, the contributions of ISG mRNA stability, cell RNA binding proteins and cell miRNAs in reducing protein levels expressed by a subset of ISGs in WNV-infected cells will be investigated. A detailed understanding of these mechanisms will increase knowledge about the complexity of the host innate response to virus infection and about a novel viral counteraction mechanism. Delineation of the viral counteraction mechanism will provide a new cell target for the development of antiviral therapeutics that may be applicable to a broad spectrum of virus infections. PUBLIC HEALTH RELEVANCE: Many flaviviruses, such as West Nile virus, tick-borne encephalitis virus, and dengue virus, are human pathogens causing significant human morbidity and mortality in ever expanding regions of the world. No effective anti-flaviviral therapies currently exist. We propose to analyze two novel Type 1 interferon- and IRF- 3/7-independent pathways for activating host innate antiviral genes in infected cells and a novel mechanism of viral counteraction of host innate responses recently discovered by our lab. A detailed understanding of these mechanisms will increase knowledge about the host innate response to virus infection and viral virulence.