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

项目摘要 右侧开放阅读框激酶3（RIOK 3）是非典型蛋白激酶中研究不足的成员 家庭的功能还没有完全确立。美国国立卫生研究院照亮了可药用基因组/ 灯塔资源，RIOK 3目前只有八个出版物，八个可能的蛋白质相互作用伙伴， 和一个小分子配体。我们最近意外地发现，RIOK 3在 抵抗病毒感染的细胞防御本文提出的研究目标是了解 RIOK 3介导细胞免疫防御，并深入了解这种激酶如何靶向于 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）是一种新兴的人类和动物病原体，可引起严重疾病 以及人类和家畜的死亡。目前还没有经过证实的、许可的治疗方法或疫苗。 RVFV感染。我们发现，在感染RVFV后，哺乳动物细胞显著上调 RIOK 3的转录。此外，感染后不久，RIOK 3 mRNA加工的新模式， 发生，导致丰富的选择性剪接的RIOK 3 mRNA，其编码缺乏的截短肽， 推定的RIOK 3激酶结构域。初步实验表明，RIOK 3的siRNA敲低 增强的病毒复制和RIOK 3的过表达减少了病毒复制，表明 RIOK 3对RVFV复制起拮抗作用。我们还观察到， 在RIOK 3已被敲除的细胞中，病毒感染减少，但这些的机制 效果不明。我们假设RIOK 3的激酶活性是启动细胞免疫的关键。 它是一个信号级联的重要组成部分，最终导致感染。 抗病毒状态 该研究的目的是研究全长的功能和分子间的相互作用， 和病毒诱导的RIOK 3的截短同种型。使用RIOK 3敲除和CRISPR敲除细胞，我们 将在转染的细胞中表达RIOK 3的全长、截短和激酶无效形式。我们将测量 病毒繁殖作为不同亚型的函数，我们将测量细胞的抗病毒反应， 通过已知的响应标记。我们还将利用在Pharos中鉴定的RIOK 3的潜在抑制剂， 激酶组筛选以评估对炎症/抗病毒标志物的影响。与此同时，我们将调查 使用免疫共沉淀实验和磷酸蛋白质组学研究RIOK 3的相互作用伙伴。的 这项试点工作的结果将成为NIH更大规模申请的基础，该申请将描述一种潜在的新的 应用于抗病毒治疗和/或调节细胞内 炎症反应。

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.