Innate immune response is the first line of host defense against infection. MDA5 is one of the RIG-I-like receptors (RLRs), which are cytoplasmic viral RNA sensors and triggers the rapid innate immune response after viral infection. MDA5 recognizes relatively long dsRNA, leading to the multimer formation of MDA5 along the dsRNA required for activating the MAVS adaptor. In response to viral infection, MDA5 is activated by protein phosphatese 1α / γ-mediated dephosphorylation of the MDA5 CARDs.
Thus, the preceding phosphorylation of de novo MDA5 prevents excessive activation of MDA5. We previously identified RIO kinase 3 (RIOK3) as a kinase targeting MDA5 using yeast two-hybrid screening. Our studies revealed that RIOK3 phosphorylates Ser-828 in MDA5 C-terminal region. Moreover, we found that RIOK3-mediated phosphorylation interferes with MDA5 multimer formation and suppresses its signaling. These findings suggest that switching between phosphorylation and dephosphorylation in both N-terminal and C-terminal region is a key factor in MDA5 functional regulation.
To examine the role of RIOK3 in vivo, we recently established RIOK3 knockout mice with CRISPR-Cas9 systems. In mouse embolic fibroblast (MEF) and bone marrow-derived macrophage (BMM) but not bone marrow-derived dendritic cell (BMDC), knockout of RIOK3 increased type I IFN expression in response to EMCV infection or poly(I:C) stimulation, which are recognized by MDA5. On the other hand, the innate immune response against VSV infection or LPS, dsDNA, 2’-3’-cGAMP stimulation was comparable between wild and RIOK3-/- MEFs. Taken together, these data suggest that RIOK3 works as a negative regulator of MDA5-mediated signaling in cell type specific manner, but barely affects RIG-I-, TLR4-, or STING-mediated signaling. Hereafter, we are going to investigate the function of RIOK3 in viral infection in vivo using an EMCV infection model.