Alternative regulation of ISGs in WNV-infected cells

WNV 感染细胞中 ISG 的替代调节

• 批准号: 8500175
• 负责人: Margo A Brinton
• 金额: $ 17.39万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8500175
• 关键词: 3' 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 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

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.

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。目前尚无有效的抗病毒疗法可用于治疗黄病毒感染个体，并且黄病毒引起的发病机制尚不完全清楚。已经发现了干扰素刺激基因 (ISG) 子集存在两种 1 型 IFN 和 IRF-3/7 独立细胞“备用”上调途径的证据，以及通过抗病毒 ISG 子集抑制蛋白质产生的新型病毒介导的对抗机制的证据。建议进行初步探索性研究来定义所涉及的机制。具体目标集中于初步了解：(1) 在 WNV 感染细胞中，在不存在典型的 I 型干扰素信号通路的情况下，感染后 12 小时，负责 IRF-3/7 独立 ISG 子集上调的机制，以及在 WNV 感染细胞中表现出延迟激活的 ISG 的第二子集所需的不同调节机制； (2) WNV 感染细胞中第二组 ISG 的蛋白质表达受到抑制的机制。在目标 1 下，我们将在 WNV 感染细胞中快速上调和延迟上调的模型 ISG 启动子中绘制与干扰素无关的 ISG 上调相关的关键转录因子结合位点 (TFBS)。所绘制的 TFBS 的功能相关性将通过诱变以及体外和体内 DNA-蛋白质相互作用测定进行测试。与映射的 TFBS 结合的转录因子将通过 DNA Pull-down 获得的蛋白质的 MassSpec 测序来鉴定。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 子集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对宿主对病毒感染的先天反应的复杂性以及新的病毒对抗机制的了解。病毒对抗机制的描述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。