在哺乳动物中，右开放阅读框激酶3（RIOK3）与癌症发展和免疫调节有关。为了探索鱼类中RIOK3在抗病毒天然免疫中的作用，本研究克隆并鉴定了黑鲩（Mylopharyngodon piceus）中RIOK3的同源物(bcRIOK3)。序列分析表明，bcRIOK3在脊椎动物中具有保守性。bcRIOK3的转录在宿主细胞对鲤鱼春季病毒（SVCV）、聚核苷酸（poly (I:C)）和脂多糖（LPS）的刺激下变化。免疫印迹（IB）和免疫荧光（IF）实验表明，bcRIOK3是一个分子量约为60 kDa的细胞质蛋白。有趣的是，bcRIOK3沉默导致IFNa、IFNb和Viperin基因的基础mRNA水平降低；然而，在病毒感染后，这些基因的mRNA水平明显增加，并增强了宿主对SVCV的抗性。与哺乳动物的同源物类似，bcRIOK3在EPC细胞中的过表达对黑鲩MDA5（bcMDA5）介导的干扰素启动子转录和抗病毒活性有显著的抑制作用。共免疫沉淀和免疫荧光实验证实了bcRIOK3和bcMDA5之间的相互作用。进一步分析揭示了bcRIOK3增强了bcMDA5的K48链连接泛素化和蛋白酶体依赖性降解，并削弱了在poly(I:C)刺激下bcMDA5的寡聚化。总之，我们的数据表明，在黑鲩中，RIOK3通过促进MDA5的降解抑制了其介导的干扰素信号传导，这为鱼类中干扰素信号传导的调控提供了新的见解。版权所有 © 2023 Elsevier Ltd.

In mammals, right open reading frame kinase 3 (RIOK3) is related with cancer development and immune regulation. To explore the role of teleost RIOK3 in the antiviral innate immunity, the homolog of RIOK3 (bcRIOK3) from black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. Sequence analysis revealed that bcRIOK3 is conserved in vertebrates. The transcription of bcRIOK3 varied in host cells in response to the stimulation of spring viremia of carp virus (SVCV), poly (I:C), and LPS. Immunoblotting (IB) and immunofluorescence (IF) assays identified bcRIOK3 as a cytoplasmic protein with a molecular weight of ∼60 kDa. It was interesting that bcRIOK3 knockdown led to the decreased basal mRNA levels of IFNa, IFNb and Viperin; however, triggered obviously higher mRNA levels of the above genes after viral infection and enhanced host resistance to SVCV. Like its mammalian counterpart, bcRIOK3 overexpression in EPC cells showed a significant inhibitory effect on black carp MDA5 (bcMDA5)-mediated transcription of interferon promoters and antiviral activity. Co-immunoprecipitation and immunofluorescent assays identified the association between bcRIOK3 and bcMDA5. Further analysis revealed that bcRIOK3 enhanced the K48-linked ubiquitination and proteasome-dependent degradation of bcMDA5, and it weakened the oligomerization of bcMDA5 under poly(I:C) stimulation. In summary, our data conclude that RIOK3 dampens MDA5-mediated IFN signaling by promoting its degradation in black carp, which provide new insights into the regulation of IFN signaling in teleost.Copyright © 2023. Published by Elsevier Ltd.