Date Published: August 8, 2013
Publisher: Public Library of Science
Author(s): Hiroyuki Oshiumi, Moeko Miyashita, Misako Matsumoto, Tsukasa Seya, Michael Gale.
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.
The innate immune system is essential for controlling virus infections, and several viruses have evolved strategies to evade host innate immune responses. Cytoplasmic viral RNA is recognized by RIG-I-like receptors, including RIG-I and MDA5 , . The RIG-I protein comprises N-terminal Caspase Activation and Recruitment Domains (CARDs), a central RNA helicase domain, and a C-terminal Repressor domain (RD) . RD consists of C-terminal RNA binding domain (CTD) and a bridging domain between CTD and helicase . RIG-I CARDs are essential for triggering the signal that induces type I interferon (IFN). In resting cells, RIG-I RD represses its CARDs signaling . After viral infection, RIG-I RD recognizes 5′-triphosphate double-stranded RNA (dsRNA), which results in a conformational change in the RIG-I protein . This conformational change leads to the release of RD autorepression of CARDs, after which CARDs associate with an IPS-1 adaptor molecule (also called MAVS, Cardif, and VISA) localized at the outer membrane of mitochondria , , , , . IPS-1 activates downstream factors such as TBK1, IKK-ε, and NEMO , , . NEMO forms a complex with TBK1 and IKK-ε and has a polyubiquitin binding region . These protein kinases are essential for activating transcription factors such as IRF-3 to induce type I IFN production .
RIG-I activation is regulated by two ubiquitin ligases Riplet and TRIM25 , . The two ubiquitin ligases are essential for RIG-I activation , , however the functional difference had been unclear. It is known that TRIM25 is essential for RIG-I oligomerization and association with IPS-1 adaptor molecule , . Here, we demonstrated that Riplet was essential for the release of RIG-I RD autorepression of its CARDs, which resulted in the association with TRIM25. This functional difference explained the reason why RIG-I requires the two ubiquitin ligases for triggering the signal.