Alternative regulation of ISGs in WNV-infected cells

WNV 感染细胞中 ISG 的替代调节

基本信息

批准号：
8385421
负责人：
Margo A Brinton
金额：
$ 22.13万
依托单位：
GEORGIA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8385421
关键词：
3&apos Untranslated Regions ATF2 gene Affinity Amino Acid Sequence Antiviral Agents Antiviral Therapy Apoptosis Binding Binding Sites Biological Assay Cell Nucleus Cells Complex DNA DNA-Protein Interaction Data Dengue Virus Detection Development Down-Regulation EMSA Flavivirus Flavivirus Infections Genes Genetic Transcription Human IFNAR1 gene IRF1 gene ISGF3G protein In Vitro Individual Infection Interferon Type I Interferon-beta Interferons JAK1 gene JUN gene Japanese Encephalitis Viruses Japanese encephalitis virus Knockout Mice Knowledge Luciferases MAPK14 gene Maps Mass Spectrum Analysis Measures Mediating Messenger RNA Modeling Mus Mutagenesis Mutation NF-kappa B Nuclear Translocation Pathogenesis Pathway interactions Peptide Sequence Determination Phosphotransferases Polyribosomes Production Promoter Regions Proteins RNA RNA-Binding Proteins Regulation Relative (related person)Reporter STAT1 gene STAT2 gene Signal Pathway Signal Transduction Site Surface Testing Therapeutic Tick-Borne Encephalitis Virus Tick-Borne Encephalitis Viruses Time Transcriptional Regulation Translating Translational Regulation Translations Up-Regulation Viral Virulence Virus Diseases West Nile virus biological adaptation to stress crosslink human CASP4 protein human morbidity human mortality in vivo induced pluripotent stem cell interferon alpha receptor interferon alpha-beta receptor mRNA Expression mRNA Stability novel novel virus pathogen promoter protein expression response sensor transcription factor viral RNA

项目摘要

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.

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，tick虫传播的脑炎病毒和登革热病毒，都是重要的人类病原体。目前尚无有效的抗病毒疗法用于治疗黄病毒感染和黄酮病毒诱导的发病机理的患者。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，用于干扰素刺激基因（ISG）的子集以及针对抗病毒ISGS子集抑制蛋白质产生蛋白质产生的新型病毒介导的反应机制。提出了最初的探索性研究来定义所涉及的机制。具体的目的集中在获得以下方面的初步理解：（1）在感染后，在感染后12小时内将IRF-3/7独立ISG子集的上调的机制在缺乏WNV感染细胞中的I型I型干扰素信号通路后进行12小时，并显示出不同的监管机制，以显示二级激活的ISG激活机构。（2）在WNV感染的细胞中抑制了ISG的第二个子集蛋白表达的机制。在AIM 1下，我们将绘制与ISG启动子中ISG的非依赖性依赖性上调的关键转录因子结合位点（TFBS），这些模型ISG迅速上调的启动子和在WNV受感染细胞中显示出上调延迟上调的启动子。映射的TFBS的功能相关性将通过诱变，体外和体内DNA-蛋白质相互作用测定测试。通过大量测序通过DNA下拉获得的蛋白质测序，将确定与映射TFBS结合的转录因子。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WNV感染细胞中ISGs子集表达的蛋白水平中的贡献。对这些机制的详细理解将增加对宿主对病毒感染的先天反应以及新型病毒反作用机制的复杂性的知识。病毒反作用机制的描述将为开发抗病毒治疗剂提供新的细胞目标广泛的病毒感染。公共卫生相关性：许多黄素病毒，例如西尼罗河病毒，tick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引起了重大的人类发病率和死亡率。目前尚无有效的抗氟病毒疗法。我们建议分析两种新型1型干扰素和IRF-3/7独立途径，用于激活感染细胞中的宿主先天抗病毒基因，以及我们实验室最近发现的宿主先天反应的病毒反应的新型机制。对这些机制的详细理解将增加对宿主对病毒感染和病毒毒力的先天反应的了解。