Abstract
MDA5 belongs to the RIG-I-like receptor family and plays a non-redundant role in recognizing cytoplasmic viral RNA to induce the production of type I IFNs. Upon RNA ligand stimulation, we observed the redistribution of MDA5 from the cytosol to mitochondrial membrane fractions. However, the molecular mechanisms of MDA5 activation remain less understood. Here we show that 14-3-3η is an essential accessory protein for MDA5-dependent type I IFN induction. We found that several 14-3-3 isoforms may interact with MDA5 through the CARDs (N-MDA5), but 14-3-3η was the only isoform that could enhance MDA5-dependent IFNβ promoter activities in a dose-dependent manner. Knock-down of 14-3-3η in Huh7 cells impaired and delayed the kinetics of MDA5 oligomerization, which is a critical step for MDA5 activation. Consequently, the MDA5-dependent IFNβ promoter activities as well as IFNβ mRNA expression level were also decreased in the 14-3-3η knocked-down cells. We also demonstrated that 14-3-3η is essential in boosting the activation of MDA5-dependent antiviral innate immunity during viral infections. In conclusion, our results uncover a novel function of 14-3-3η to promote the MDA5-dependent IFNβ induction pathway by reducing the immunostimulatory potential of viral dsRNA within MDA5 activation signaling pathway.
Highlights
Among the RIG-I-like Receptor (RLR) family, RIG-I and MDA5 share a number of structural similarities, and both of them include three distinct domains
We have previously identified that activated RIG-I will redistribute to the mitochondrion-associated membrane (MAM) through interaction with mitochondrial chaperone protein 14-3-3ε via the caspase activation and recruitment domains (CARDs) of RIG-I [7]
Huh7 Non-Targeting Vector (NTV) cells, Huh7 RIG-I knock-down (K/D) cells, Huh7 14-3-3η K/D cells were first transfected with pSUPER.retro.shRNA plasmids (Oligoengine) which contain the shRNA sequences for targeting genes respectively and subsequently selected and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1 μg/mL puromycin
Summary
Among the RIG-I-like Receptor (RLR) family, RIG-I and MDA5 share a number of structural similarities, and both of them include three distinct domains. The interaction between MDA5 to the long dsRNA will cooperatively form a tandem MDA5 filament along the dsRNA, and the CARDs of MDA5 will oligomerize and interact with MAVS to trigger antiviral signaling pathway [12]. This signaling pathway leads to the phosphorylation and activation of transcription factors, such as interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) to induce interferon β (IFNβ) and a set of other antiviral genes to restrict virus replication [13,14,15]
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