Phosphorylation-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.
Gack
Michaela U
MU
Department of Microbiology and Molecular Genetics, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive, Box 9102, Southborough, MA 01772-9102, USA. michaela_gack@hms.harvard.edu
Nistal-Villán
Estanislao
E
Inn
Kyung-Soo
KS
García-Sastre
Adolfo
A
Jung
Jae U
JU
D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D015398:Signal Transduction; D014157:Transcription Factors / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism; D014777:Virus Diseases / Q000517:prevention & control*
D048868:Adaptor Proteins, Signal Transducing; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D025801:Ubiquitin; C512612:MAVS protein, mouse; C490034:Trim25 protein, mouse; C503356:Ddx58 protein, mouse