A Distinct Role of Riplet-Mediated K63-Linked Polyubiquitination of the RIG-I Repressor Domain in Human Antiviral Innate Immune Responses

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. © 2013 oshiumi et al.