Black carp RIOK3 suppresses MDA5-mediated IFN signaling in the antiviral innate immunity

Abstract

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.