Phosphorylation-Dependent Feedback Inhibition of RIG-I by DAPK1 Identified by Kinome-wide siRNA Screening

Abstract

Cell-autonomous induction of type I interferon must be stringently regulated. Rapid induction is key to control virus infection, whereas proper limitation of signaling is essential to prevent immunopathology and autoimmune disease. Using unbiased kinome-wide RNAi screening followed by thorough validation, we identified 22 factors that regulate RIG-I/IRF3 signaling activity. We describe a negative-feedback mechanism targeting RIG-I activity, which is mediated by death associated protein kinase 1 (DAPK1). RIG-I signaling triggers DAPK1 kinase activation, and active DAPK1 potently inhibits RIG-I stimulated IRF3 activity and interferon-beta production. DAPK1 phosphorylates RIG-I in vitro at previously reported as well as other sites that limit 5'ppp-dsRNA sensing and virtually abrogate RIG-I activation.

Assembly of the WHIP-TRIM14-PPP6C Mitochondrial Complex Promotes RIG-I-Mediated Antiviral Signaling

“…These data suggest that PPP6C acts as a positive feedback regulator by dephosphorylating RIG-I. A recent study revealed a negative feedback mechanism of DAPK1 in RIG-I phosphorylation after RIG-I activation (Willemsen et al, 2017). To demonstrate a potential feedback role of PPP6C, we examined the RIG-I S8 phosphorylation status at early and later time points during RIG-I activation in WT, PP1 KO, and PPP6C KO cells infected with VSV.…”

“…Serving as an auto-inhibitory mechanism, the N terminus of RIG-I is highly phosphorylated by protein kinase C (PKC) (Maharaj et al, 2012). RIG-I can be activated by PP1a or PP1g through dephosphorylation after RNA binding, which can be suppressed again through feedback-induced phosphorylation by DAPK1 (Willemsen et al, 2017). Thus, the S8 and T170 phosphorylation status is regulated by dynamic regulatory mechanisms.…”

Reciprocal Effects of Fibroblast Growth Factor Receptor Signaling on Dengue Virus Replication and Virion Production

“…The primary screening was performed by reverse transfection cell arrays with a human siRNA kinome library as described elsewhere (Erfle et al, 2007;Willemsen et al, 2017;Reiss et al, 2011). This library targets all known and predicted 719 human kinases with three individual siRNAs per gene (Silencerâ Human Kinase siRNA Library V3, AM80010V3; Ambion, ThermoFischer Scientific).…”

Gene Expression Profiling of Different Huh7 Variants Reveals Novel Hepatitis C Virus Host Factors

“…RNA isolation from cell lysates, reverse transcription and quantitative real-time PCR were performed as described in [59]. Briefly, 1 µg of isolated whole cell RNA was reverse-transcribed with random hexamer primers and gene expression levels were assessed with gene-specific primers and SYBR ® Green (Bio-Rad, Feldkirchen, Germany).…”

“…Cell lines stably overexpressing putative HCV host factors were generated as described in [59]. Briefly, lentiviral particles were produced in HEK293T cells [66] by calcium phosphate transfection and used for transduction of target cells.…”