Regulation Of Cellular Antiviral Response By GSK3β And Fox01

The innate immune system contributes the first line of host to defense against viral infections. During infection, many viral pathogen-associated molecular patterns (PAMPs) such as double-stranded RNA (dsRNA) are generated and recognized by host pattern-recognition receptors (PRRs) which activate production of type I IFNs and cellular antiviral response.One family of such PRRs is called RIG-like receptors (RLRs), which bind to viral dsRNA and undergo conformational changes. VISA (virus-induced signaling adaptor), a key adaptor protein located on mitochondria, is recruited to RLRs to form prion-like aggregates and mediate signal transduction. VISA interacts with another adaptor MITA (mediator of IRF3activation) and promotes phosphorylation of IRF3by TBK1. The phosphorylated IRF3forms dimer and enters into nucleus. VISA also recruits TRAF6to activate the IKK complex, which results in phosphorylation and degradation of IκBα, thereby releasing NF-κB into nucleus. The activated IRF3and NF-κB bind to the promoter of IFNB1gene and collaborate to initiate its transcription.Here, we identified GSK3β (glycogen synthase kinase3β) as an essential regulator for virus-triggered IFN-β induction. Overexpression of GSK3β potentiated virus-triggered IRF3activation and1FNB1transcription. While knockdown or delete Gsk3b impaired virus-triggered IRF3activation and IFN-β induction, as well as cellular antiviral response. Reconstitution Gsk3b-/-MEFs with GSK3β restored the antiviral response. GSK3β interacted with TBK1in a virus infection-dependent manner, and promoted TBK1self-association and self-phosphorylation on Serl72, which is critical for IRF3activation and IFN-β induction. It has been reported that the kinase activity of GSK3β is important for its function in regulation of various signaling pathway. However, in our experiments, we found that both the kinase inactive mutant GSK3β (K85A) and constitutive active mutant GSK3P (S9A) could similarly potentiate virus-induced production of type I IFNs. Furthermore, reconstitution of GSK3p (K85A) and GSK3p (S9A) into Gsk3b-/-MEFs restored virus-induced production of type I IFNs. Our results thus suggest that the effect of GSK3P on virus-triggered IFN-β induction is independent on the GSK3P kinase activity.Although type I IFNs are critical for immune response, overproduction of IFNs causes a pathogenic immune response, which is harmful to host, even leading to host death. Therefore, the production of type I IFNs should be tightly controlled to avoid excessive immune response.In this study, we identified FoxOl (forkhead box protein01) as a negative regulator of virus-triggered IFNs induction. Overexpression of FoxO1inhibited virus-triggered ISRE activation, IFN-P induction as well as cellular antiviral response, whereas knockdown of FoxO1had opposite effect. FoxOl interacted with IRF3in the cytosol in a virus infection-dependent manner. Furthermore, FoxO1promoted K48-linked poly-ubiquitination and degradation of IRF3which is independent of the known E3ligase RBCK1-and RAUL-mediate the IRF3poly-ubiquitination and degradation.As a summary, this study has clearly shown that innate antiviral response are tightly regulated by GSK3P and FoxO1. GSK3β interacts with TBK1in a virus infection-dependent manner, and promotes TBK1self-association and self-phosphorylation which is critical for virus triggered type I IFNs induction. Whereas FoxO1interacts with IRF3in a virus infection-dependent manner, and promote K48-linked poly-ubiquitination and degradation of IRF3, thereby inhibiting virus triggered IFNs induction. This study contributes to the elucidation of complicated molecular mechanisms of cellular antiviral responses, and provides new insight into the molecular mechanisms for the innate immune response.