Alternative regulation of ISGs in WNV-infected cells

描述(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、森林脑炎病毒和登革热病毒，都是重要的人类病原体。目前还没有有效的抗病毒疗法来治疗黄病毒感染的个体，而且黄病毒诱导的发病机制还不完全清楚。有证据表明，干扰素刺激基因(ISG)的亚组存在两个1型干扰素和IRF-3/7独立的细胞“备份”上调途径，以及一种新的病毒介导的对抗机制，该机制旨在通过抗病毒ISG亚组抑制蛋白质的产生。提出了初步的探索性研究，以确定所涉及的机制。具体目标集中在获得以下方面的初步了解：(1)在西尼罗河病毒感染的细胞中缺乏规范的I型干扰素信号通路的情况下，感染后12小时内IRF-3/7非依赖的ISGs子集上调的机制，以及第二亚组ISGs在西尼罗河病毒感染的细胞中显示延迟激活所需的不同调控机制；以及(2)在西尼罗河病毒感染的细胞中第二ISG子集的蛋白表达被抑制的机制。在目标1下，我们将在西尼罗河病毒感染细胞中快速上调和延迟上调的模型ISGs的启动子中定位涉及干扰素非依赖性上调ISGs的关键转录因子结合位点(TFBs)。将通过突变以及体外和体内DNA-蛋白质相互作用试验来检验定位的TFBS的功能相关性。通过对DNA下拉获得的蛋白质进行MassSpec测序，将识别与映射的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miRNAs在降低西尼罗河病毒感染细胞中ISG亚群表达的蛋白质水平方面的贡献。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一种新的病毒对抗机制的知识。病毒对抗机制的阐明将为抗病毒治疗药物的开发提供新的细胞靶点。 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、壁虱传播的脑炎病毒和登革热病毒，都是人类病原体，在世界上不断扩大的地区造成重大的人类发病率和死亡率。目前还没有有效的抗黄病毒疗法。我们建议分析两条新的1型干扰素和IRF-3/7非依赖的途径，以激活感染细胞中宿主固有的抗病毒基因，以及我们实验室最近发现的一种新的病毒对抗宿主固有反应的机制。对这些机制的详细了解将增加关于宿主对病毒感染和病毒毒力的先天反应的知识。

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.