Role of the atypical protein kinase RIOK3 in the cellular antiviral response

非典型蛋白激酶 RIOK3 在细胞抗病毒反应中的作用

• 批准号: 9813704
• 负责人: J. Stephen Lodmell
• 金额: $ 14.28万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-05 至 2022-09-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9813704
• 关键词: Affect; Affinity; Africa; Alternative Splicing; Americas; Animals; Anti-inflammatory; Antiviral Agents; Antiviral Response; Antiviral Therapy; Appearance; Autoimmune Diseases; Binding; Biological Assay; Biological Process; Blindness; Cell Culture Techniques; Cell Line; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Code; Complementary DNA; Coupled; Culicidae; Data; Disease; Europe; Family; Genetic Transcription; Genome; Goals; Human; Immune; Immune response; Infection; Inflammatory; Inflammatory Response; Innate Immune Response; Interferon-beta; Invaded; Knock-out; Lead; Length; Ligands; Livestock; Mammalian Cell; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Mutate; Nonsense-Mediated Decay; Open Reading Frames; Outcome; Pathway interactions; Pattern; Peptides; Phosphotransferases; Plaque Assay; Plasmids; Play; Production; Protein Binding Domain; Protein Isoforms; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Publications; Quantitative Reverse Transcriptase PCR; RNA Splicing; Reporter Genes; Research; Resources; Rift Valley fever virus; Role; Signal Transduction; Small Interfering RNA; Testing; Transcript; Translating; United States National Institutes of Health; Vaccines; Viral; Viral Genome; Viral Hemorrhagic Fevers; Virus; Virus Diseases; Virus Replication; Western Blotting; Work; base; climate change; design; experimental study; expression vector; inhibitor/antagonist; insight; kinase inhibitor; knock-down; liver injury; member; mosquito-borne; mutant; novel therapeutic intervention; online resource; overexpression; pathogen; phosphoproteomics; protein protein interaction; response; response biomarker; small molecule; success; tool; treatment strategy

Project Summary The RIght Open reading frame Kinase 3, or RIOK3, is an understudied member of the atypical protein kinase family whose function has not been firmly established. On the NIH Illuminating the Druggable Genome/ Pharos resource, RIOK3 currently boasts only eight publications, eight possible protein interaction partners, and one small molecule ligand. We recently found, quite unexpectedly, that RIOK3 plays an important role in the cellular defense against viral infection. The goal of the research proposed here is to understand how RIOK3 mediates cellular immune defenses and to gain insight into how this kinase could be targeted for antiviral and/or anti-inflammatory therapies. Results will be made available on the IDG/Pharos web resources. Rift Valley fever virus (RVFV) is an emerging human and animal pathogen that can cause serious disease and death in humans and domestic livestock. There is currently no proven, licensed treatment or vaccine for RVFV infections. We found that upon infection with RVFV, mammalian cells dramatically upregulate transcription of RIOK3. Furthermore, shortly after infection, a new pattern of RIOK3 mRNA processing occurs, resulting in abundant alternatively spliced RIOK3 mRNAs that code for a truncated peptide lacking the putative RIOK3 kinase domain. Preliminary experiments showed that siRNA knockdown of RIOK3 enhanced viral replication, and overexpression of RIOK3 diminished viral replication, demonstrating that RIOK3 plays an antagonistic role to RVFV replication. We also observed that production of interferon-β after infection with virus is diminished in cells in which RIOK3 has been knocked out, but the mechanism for these effects is unknown. We hypothesize that RIOK3's kinase activity is key to mounting a cellular immune response to infection and that it is an essential component to a signaling cascade that culminates in an antiviral state. The goal of the proposed research is to investigate the function and molecular interactions of the full-length and virally-induced truncated isoform of RIOK3. Using RIOK3 knockdown and CRISPR knockout cells, we will express the full length, truncated, and kinase-null versions of RIOK3 in transfected cells. We will measure virus propagation as a function of the different isoforms, and we will measure the cellular antiviral response via known response markers. We will also make use of potential inhibitors of RIOK3 identified in Pharos from kinome screens to assess effects on inflammatory/antiviral markers. In parallel, we will investigate the interacting partners of RIOK3 using co-immunoprecipitation experiments and phosphoproteomics. The results of this pilot work will form the basis for a larger NIH application that will describe a potential new druggable kinase target with applications to antiviral therapy and/or the modulation of the cellular inflammatory response.

项目概要 RIight 开放阅读框激酶 3（或 RIOK3）是非典型蛋白激酶中一个尚未得到研究的成员 NIH 阐明可药物基因组/的功能尚未确定的家族。 Pharos 资源，RIOK3 目前仅拥有八个出版物，八个可能的蛋白质相互作用伙伴， 我们最近出乎意料地发现，RIOK3 在其中发挥着重要作用。 这里提出的研究的目的是了解细胞如何防御病毒感染。 RIOK3 介导细胞免疫防御并深入了解该激酶如何靶向 抗病毒和/或抗炎治疗结果将在 IDG/Pharos 网站上公布。 资源。 裂谷热病毒（RVFV）是一种新出现的人类和动物病原体，可引起严重疾病 以及人类和家畜死亡 目前尚无经过验证的、获得许可的治疗方法或疫苗。 我们发现，感染 RVFV 后，哺乳动物细胞的表达显着上调。 此外，感染后不久，RIOK3 mRNA 加工的新模式。 发生，导致产生丰富的选择性剪接的 RIOK3 mRNA，编码缺乏的截短肽 初步实验表明 RIOK3 的 siRNA 敲低。 增强病毒复制，而 RIOK3 的过度表达则减少病毒复制，这表明 RIOK3对RVFV复制发挥拮抗作用，我们还观察到干扰素-β的产生。 在 RIOK3 被敲除的细胞中，病毒感染减少，但这些机制 我们关心的是 RIOK3 的激酶活性是建立细胞免疫的关键。 对感染的反应，并且它是信号级联的重要组成部分，最终导致 抗病毒状态。 拟议研究的目标是研究全长的功能和分子相互作用 和病毒诱导的 RIOK3 截短亚型，我们使用 RIOK3 敲低和 CRISPR 敲除细胞。 将在转染细胞中表达 RIOK3 的全长、截短和激酶无效版本。 病毒传播作为不同亚型的函数，我们将测量细胞抗病毒反应 通过已知的反应标记，我们还将利用 Pharos 中鉴定的潜在 RIOK3 抑制剂。 同时，我们将研究激酶组筛选以评估对炎症/抗病毒标记物的影响。 使用免疫共沉淀实验和磷酸蛋白质组学研究 RIOK3 的相互作用伙伴。 这项试点工作的结果将构成 NIH 更大应用的基础，该应用将描述潜在的新方法 可药物激酶靶标应用于抗病毒治疗和/或细胞调节 炎症反应。