Abstract
The innate immune system is essential for controlling viral infections, but several viruses have evolved strategies to escape innate immunity. RIG-I is a cytoplasmic viral RNA sensor that triggers the signal to induce type I interferon production in response to viral infection. RIG-I activation is regulated by the K63-linked polyubiquitin chain mediated by Riplet and TRIM25 ubiquitin ligases. TRIM25 is required for RIG-I oligomerization and interaction with the IPS-1 adaptor molecule. A knockout study revealed that Riplet was essential for RIG-I activation. However the molecular mechanism underlying RIG-I activation by Riplet remains unclear, and the functional differences between Riplet and TRIM25 are also unknown. A genetic study and a pull-down assay indicated that Riplet was dispensable for RIG-I RNA binding activity but required for TRIM25 to activate RIG-I. Mutational analysis demonstrated that Lys-788 within the RIG-I repressor domain was critical for Riplet-mediated K63-linked polyubiquitination and that Riplet was required for the release of RIG-I autorepression of its N-terminal CARDs, which leads to the association of RIG-I with TRIM25 ubiquitin ligase and TBK1 protein kinase. Our data indicate that Riplet is a prerequisite for TRIM25 to activate RIG-I signaling. We investigated the biological importance of this mechanism in human cells and found that hepatitis C virus (HCV) abrogated this mechanism. Interestingly, HCV NS3-4A proteases targeted the Riplet protein and abrogated endogenous RIG-I polyubiquitination and association with TRIM25 and TBK1, emphasizing the biological importance of this mechanism in human antiviral innate immunity. In conclusion, our results establish that Riplet-mediated K63-linked polyubiquitination released RIG-I RD autorepression, which allowed the access of positive factors to the RIG-I protein.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Substitution
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Animals
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Carrier Proteins / genetics
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Carrier Proteins / immunology
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Carrier Proteins / metabolism*
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Chlorocebus aethiops
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DEAD Box Protein 58
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DEAD-box RNA Helicases / genetics
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DEAD-box RNA Helicases / immunology
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DEAD-box RNA Helicases / metabolism*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / immunology
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DNA-Binding Proteins / metabolism
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HEK293 Cells
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Hep G2 Cells
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Hepacivirus / genetics
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Hepacivirus / immunology
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Hepacivirus / metabolism*
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Hepatitis C / genetics
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Hepatitis C / immunology
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Hepatitis C / metabolism*
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Humans
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Immunity, Innate*
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Intracellular Signaling Peptides and Proteins
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Mice
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Mice, Knockout
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Mutation, Missense
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Protein Structure, Tertiary
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Receptors, Immunologic
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Transcription Factors / genetics
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Transcription Factors / immunology
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Transcription Factors / metabolism
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Tripartite Motif Proteins
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / immunology
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Ubiquitin-Protein Ligases / metabolism
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Ubiquitination / physiology*
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Vero Cells
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Viral Nonstructural Proteins / genetics
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Viral Nonstructural Proteins / immunology
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Viral Nonstructural Proteins / metabolism
Substances
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Carrier Proteins
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DNA-Binding Proteins
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Intracellular Signaling Peptides and Proteins
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NS3 protein, hepatitis C virus
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NS4A cofactor peptide, Hepatitis C virus
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Receptors, Immunologic
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Transcription Factors
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Trim25 protein, mouse
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Tripartite Motif Proteins
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Viral Nonstructural Proteins
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RNF135 protein, human
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Rnf135 protein, mouse
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TRIM25 protein, human
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Ubiquitin-Protein Ligases
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RIGI protein, human
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Ddx58 protein, mouse
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DEAD Box Protein 58
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DEAD-box RNA Helicases
Grants and funding
This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan, and the Ministry of Health Labour, and Welfare of Japan, Kato Memorial Bioscience Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.