280: Essential role of the phosphatase PP1 in the RIG-I and MDA5 mediated antiviral interferon response

Innate sensing of viral infection and the subsequent activation of signaling pathways are essential for an effective antiviral immune response. Retinoic acid-inducible gene-I (RIG-I; also called DDX58) and melanoma differentiation-associated gene 5 (MDA5; also known as IFIH1) have emerged as key cytosolic sensors for the detection of RNA viruses. Upon viral RNA binding, the N-terminal caspase recruitment domains (CARDs) of RIG-I and MDA5 initiate downstream signaling, resulting in the production of type-I interferon (IFN) and proinflammatory cytokines. Recent studies highlighted the important role of multisite posttranslational modification for controlling RIG-I antiviral activity; in contrast, the regulation of MDA5 signaling activity remains largely undetermined. Here, we show that MDA5 signal-transducing activity is regulated by a dynamic balance between phosphorylation and dephosphorylation of its N-terminal CARDs, and further identify the phosphoprotein phosphatase 1 α (PP1α) and PP1γ as essential activators of both MDA5 and RIG-I antiviral signal transduction. Using mass spectrometry and a phosphorylation state-specific MDA5 antibody, we identified that MDA5 is robustly phosphorylated at S88 under normal conditions; upon viral infection, however, MDA5 is rapidly dephosphorylated, allowing CARD-mediated downstream signaling. Employing a phosphatome RNA interference (RNAi) screen, we identified PP1 α and PP1γ as primary phosphatases responsible for the MDA5 and RIG-I CARD dephosphorylation, which leads to their activation. Depletion of endogenous PP1α and PP1γ markedly enhanced the CARD phosphorylation of the sensors MDA5 and RIG-I, and strongly reduced antiviral IFN-β production. Furthermore, cells in which PP1α and PP1γ had been silenced using specific siRNAs were impaired in their ability to induce the expression of various IFN-stimulated genes, which correlated with an enhanced RNA virus replication in these cells. Collectively, this work identifies PP1α and PP1γ as critical regulators of antiviral innate immune responses to a wide range of RNA viruses, including paramyxoviruses, orthomyxoviruses, dengue virus and picornaviruses.