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.
Highlights
The innate immune system is essential for controlling virus infections, and several viruses have evolved strategies to evade host innate immune responses
RIG-I activation is regulated by K63-linked polyubiquitin chains mediated by the ubiquitin ligases TRIM25 and Riplet; the functional difference between the two ubiquitin ligases remains unclear, and the molecular mechanism underlying Riplet-mediated RIGI activation is unknown
We revealed sequential roles of the two ubiquitin ligases during RIG-I activation and found that Riplet-mediated polyubiquitination of the RIG-I repressor domain released RIG-I autorepression of its Nterminal Caspase Activation and Recruitment Domains (CARDs) responsible for triggering the signal, which resulted in an association with TRIM25 ubiquitin ligase and TBK1 protein kinase
Summary
The innate immune system is essential for controlling virus infections, and several viruses have evolved strategies to evade host innate immune responses. RIG-I RD recognizes 59-triphosphate double-stranded RNA (dsRNA), which results in a conformational change in the RIG-I protein [3]. This conformational change leads to the release of RD autorepression of CARDs, after which CARDs associate with an IPS-1 adaptor molecule ( called MAVS, Cardif, and VISA) localized at the outer membrane of mitochondria [3,5,6,7,8]. NEMO forms a complex with TBK1 and IKK-e and has a polyubiquitin binding region [12] These protein kinases are essential for activating transcription factors such as IRF-3 to induce type I IFN production [13]
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