Controls of MDA5 Activation by The 14-3-3 Protein Family

Abstract

Abstract During RNA virus infection, RIG-I like receptors (RLRs), such as Retinoic acid-inducible gene I (RIG-I) and Melanoma differentiation-associated protein 5 (MDA5), recognize cytoplasmic viral RNA and activate downstream adaptor proteins to produce type I interferon (IFN). RIG-I is known to bind short dsRNA (<200 bp) whereas MDA5 prefers to bind to dsRNA which are longer than 2000 bp. It has been reported that RIG-I can interact with TRIM25, which is the ubiquitin E3 ligase of RIG-I during viral infections, and with the chaperon protein 14-3-3ɛ, which facilitate RIG-I translocate to mitochondria. However, the molecular mechanisms of MDA5 activation remain unclear. Here we show that certain 14-3-3 isoforms are crucial for MDA5-dependent type I IFN induction. Transfection of dsRNAs in different lengths to Huh7 RIG-I knocked-down cells reveals that MDA5 may specifically binds to long dsRNA and activate IFNβ promoter activity 3 hours post-RNA transfection. Also, overexpression of full-length MDA5 or the N-terminal CARD domains may constitutively activate IFNβ promoter activity, while C-MDA5 lacking both CARD did not show any ability to signal itself. We then applied dsRNA transfection or MDA5 overexpression to assess which 14-3-3 isoform is critical for MDA5 activation. We found that certain 14-3-3 isoform may bind to full-length MDA5 and N-MDA5 shown by co-immunoprecipitation, and ectopic expression of 14-3-3 may enhance MDA5-dependent activation of IFNβ promoter. Further studies of the molecular mechanisms how 14-3-3 proteins participate and regulate in the MDA5-dependent signaling pathway will be investigated in order to define the critical steps of MDA5 activation during RNA virus infections.