biostudies-literatureGack MUNCRR NIH HHSNIAID NIH HHSNCI NIH HHS3220-9https://www.ebi.ac.uk/biostudies/studies/S-EPMC2838087biostudies-literatureUnknown84(7)Recognition of invading viruses by the host is elicited by cellular sensors which trigger signaling cascades that lead to type I interferon (IFN) gene expression. Retinoic acid-inducible gene I (RIG-I) has emerged as a key receptor for the detection of viral RNA in the cytosol, inducing IFN-mediated innate immune responses to limit viral replication through its interaction with MAVS (also called IPS-1, CARDIF, or VISA). Upon the recognition of viral RNA, the Lys-172 residue of RIG-I undergoes ubiquitination induced by tripartite motif protein 25 (TRIM25), an essential protein for antiviral signal transduction. Here we demonstrate that phosphorylation represents another regulatory mechanism for RIG-I-mediated antiviral activity. Using protein purification and mass spectrometry analysis, we identified three phosphorylation sites in the amino-terminal caspase recruitment domains (CARDs) of RIG-I. One of these residues, Thr-170, is located in close proximity to Lys-172, and we speculated that its phosphorylation may affect Lys-172 ubiquitination and functional activation of RIG-I. Indeed, a RIG-I mutant carrying a phosphomimetic Glu residue in place of Thr-170 loses TRIM25 binding, Lys-172 ubiquitination, MAVS binding, and downstream signaling ability. This suggests that phosphorylation of RIG-I at Thr-170 inhibits RIG-I-mediated antiviral signal transduction. Immunoblot analysis with a phospho-specific antibody showed that the phosphorylation of the RIG-I Thr-170 residue is present under normal conditions but rapidly declines upon viral infection. Our results indicate that Thr-170 phosphorylation and TRIM25-mediated Lys-172 ubiquitination of RIG-I functionally antagonize each other. While Thr-170 phosphorylation keeps RIG-I latent, Lys-172 ubiquitination enables RIG-I to form a stable complex with MAVS, thereby inducing IFN signal transduction.Journal of virologyPhosphorylation-mediated negative regulation of RIG-I antiviral activity.PMC2838087U19 AI083025HHSN266200700010CR01AI46954P51 RR000168R01 AI046954R01 CA082057U19AI083025R01 AI087846K26 RR000168R01 AI083355RR00168AI083355CA082057Gack MUGarcia-Sastre AJung JUNistal-Villan EInn KSfalsePhosphorylation-mediated negative regulation of RIG-I antiviral activity.Recognition of invading viruses by the host is elicited by cellular sensors which trigger signaling cascades that lead to type I interferon (IFN) gene expression. Retinoic acid-inducible gene I (RIG-I) has emerged as a key receptor for the detection of viral RNA in the cytosol, inducing IFN-mediated innate immune responses to limit viral replication through its interaction with MAVS (also called IPS-1, CARDIF, or VISA). Upon the recognition of viral RNA, the Lys-172 residue of RIG-I undergoes ubiquitination induced by tripartite motif protein 25 (TRIM25), an essential protein for antiviral signal transduction. Here we demonstrate that phosphorylation represents another regulatory mechanism for RIG-I-mediated antiviral activity. Using protein purification and mass spectrometry analysis, we identified three phosphorylation sites in the amino-terminal caspase recruitment domains (CARDs) of RIG-I. One of these residues, Thr-170, is located in close proximity to Lys-172, and we speculated that its phosphorylation may affect Lys-172 ubiquitination and functional activation of RIG-I. Indeed, a RIG-I mutant carrying a phosphomimetic Glu residue in place of Thr-170 loses TRIM25 binding, Lys-172 ubiquitination, MAVS binding, and downstream signaling ability. This suggests that phosphorylation of RIG-I at Thr-170 inhibits RIG-I-mediated antiviral signal transduction. Immunoblot analysis with a phospho-specific antibody showed that the phosphorylation of the RIG-I Thr-170 residue is present under normal conditions but rapidly declines upon viral infection. Our results indicate that Thr-170 phosphorylation and TRIM25-mediated Lys-172 ubiquitination of RIG-I functionally antagonize each other. While Thr-170 phosphorylation keeps RIG-I latent, Lys-172 ubiquitination enables RIG-I to form a stable complex with MAVS, thereby inducing IFN signal transduction.2010-01-01T00:00:00Z2010 Apr2024-11-12T14:40:38.267Z2019-03-27T00:29:21ZS-EPMC28380872007158210.1128/JVI.02241-0910.1128/jvi.02241-09