Regulation of RIG-I-mediated anti-viral innate immunity by post-translational modifications
Final Report Abstract
The two cellular helicases RIG-I and MDA5 are ubiquitously expressed and sense viral infection. Engangement of these intracellular receptors by their ligands leads to a signal transduction cascade that results in the upregulation of type I interferons (IFNs) and various cytokines. For RIG-I the ligand has been defined as partially double-stranded (ds) RNA with a free triphosphate group at the 5`end. MDA5 on the other hand was described to detect long ds RNA or even more complex RNA structures. Consequently, the two helicases recognize a different subset of viruses. Whereas RIG-I’s antiviral activity is directed against paramyxoviruses, influenza virus, vesicular stomatitis virus, and Hepatitis C virus, MDA5 primarily senses picornaviruses. In addition, it has been recently shown that MDA5 is crucial for the host defense to paramyxoviruses in vivo. Both RIG-I and MDA5 function redundantly in the detection of Dengue virus and West Nile virus. Activation and signal transduction of these molecules is tightly regulated both at the level of the helicase itself as well as downstream in the signaling cascade. For RIG-I, specifically, a complex pattern of regulatory post-translational modifications has been described so far. In uninfected cells, RIG-I is negatively regulated by phosphorylation at Serine 8 and Threonine 170 residues, keeping the molecule inactive for signaling. Dephosphorylation of RIG-I followed by ubiquitinylation is a prerequisite for the induction of a RIG-I signaling cascade. In contrast, prior to this project there was a huge gap of knowledge regarding the regulation of the second sensor MDA5. During the course of this project, we could identify that MDA5, analogous to RIG-I, is regulated by a dynamic balance between phosphorylation and dephosphorylation. Massspectrometry analyses as well as experiments with a phospho-state specific MDA5 antibody showed that MDA5 is robustly phosphorylated at Serine 88 prior to viral infection. Ligand binding leads to rapid dephosphorylation and initiation of MDA5 signaling. Mutational analyses further confirmed that MDA5 is kept in an inactive state by its phosphorylation. Employing a siRNA-based screening including siRNAs against all known human phosphatases we were able to identify Protein Phosphatase 1 (PP1) isoform α and isoform γ as the primary phosphatase for MDA5 dephosphorylation. Our screening results were verified by a complementary set of PP1 knockdown and overexpression experiments, measuring MDA5- dependent interferon-β promoter activation and secretion. Moreover, experiments with a phospho-state specific MDA5 and RIG-I antibody revealed that both sensors, MDA5 and RIG-I, are targeted by PP1 for dephosphorylation. Consequently IFN-stimulated gene induction and replication of viruses belonging to five different families, namely paramyxoviruses (SeV), rhabdoviruses (VSV), orthomyxoviruses (influenza virus), picornaviruses (EMCV) and Flaviviruses (Dengue virus) are affected by siRNA mediated knockdown of PP1α/γ. Furthermore, the identification of PP1 binding motifs in RIG-I and MDA5 as well as the functional characterization of PP1 binding-deficient mutants of both sensors support that PP1α/γ play crucial roles in RLR signaling. Interaction and confocal microscopy studies revealed that PP1α/γ are in a complex with MDA5 and RIG-I only in virus infected cells, providing evidence that the specific recruitment of the phosphatase to these sensors triggers their rapid dephosphorylation. In summary, our results give new insights into the complex regulatory network of the RIG-I and MDA5 signaling pathway, and identify PP1α and PP1γ as novel regulators of interferon induction.
Publications
- Conventional protein kinase C-α (PKC-α) and PKC-β negatively regulate RIG-I antiviral signal transduction. J Virol. 2012 Feb;86(3):1358-71. Epub 2011 Nov 23
Maharaj NP, Wies E, Stoll A, Gack MU
