Alternative regulation of ISGs in WNV-infected cells

描述（由申请人提供）：许多黄病毒，包括西尼罗河病毒（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疗法用于治疗黄病毒感染的个体，并且黄病毒诱导的发病机制尚未完全了解。已经发现了干扰素刺激基因（ISG）子集的两种1型IFN-和IRF-3/7-独立的细胞“后备”上调途径的存在以及针对抑制抗病毒ISG子集的蛋白质产生的新型病毒介导的对抗机制的证据。初步的探索性研究，建议确定所涉及的机制。具体目标集中于获得以下方面的初步理解：（1）在WNV感染的细胞中，在不存在经典I型干扰素信号传导途径的情况下，在感染后12小时，负责IRF-3/7非依赖性ISG子集上调的机制，以及负责在WNV感染的细胞中显示延迟激活的第二ISG子集所需的不同调节机制的机制;和（2）在WNV感染的细胞中抑制ISG的第二子集的蛋白质表达的机制。在目标1下，我们将在模型ISG的启动子中绘制参与ISG的干扰素非依赖性上调的关键转录因子结合位点（TFBS），所述启动子在WNV感染的细胞中快速上调和显示延迟上调的那些。将通过诱变以及体外和体内DNA-蛋白质相互作用测定来测试映射的TFBS的功能相关性。将通过对通过DNA下拉获得的蛋白质进行MassSpec测序来鉴定与映射的TFBS结合的转录因子。在目标2下，将研究ISG mRNA稳定性、细胞RNA结合蛋白和细胞miRNA在降低WNV感染细胞中ISG亚组表达的蛋白水平中的作用。对这些机制的详细了解将增加关于宿主对病毒感染的先天反应的复杂性和关于新的病毒对抗机制的知识。病毒对抗机制的阐明将为开发可能适用的抗病毒疗法提供新的细胞靶点 广泛的病毒感染。 公共卫生相关性：许多黄病毒，如西尼罗河病毒、蜱传脑炎病毒和登革热病毒，是在世界不断扩大的地区引起显著的人类发病率和死亡率的人类病原体。目前不存在有效的抗黄病毒疗法。我们建议分析两种新的1型干扰素和IRF- 3/7独立的途径激活宿主先天抗病毒基因在感染的细胞和一种新的机制，病毒抵消宿主先天反应最近发现我们的实验室。对这些机制的详细了解将增加对宿主对病毒感染和病毒毒力的先天反应的认识。

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.