The cytosolic RIG-I pathway is activated by many RNA viruses - including influenza - via viral RNA replicative intermediates that contain short hairpin dsRNA and 5¡ä triphosphate structures. We characterized natural and synthetic RIG-I agonists based on sequences from the 5¡¯UTR regions of distinct negative-strand viruses - vesicular stomatitis virus (VSV), Influenza, Rabies, Measles, and Sendai virus - and demonstrated potent stimulation of RIG-I antiviral responses at concentrations in the picomolar range. In human bronchial epithelial A549 cells, 5¡äpppRNA induced IRF3 phosphorylation and dimerization, STAT1 Tyr701 phosphorylation, as well as a >100-fold increase in the transcription of interferon stimulated genes (ISGs) and genes involved in inflammation. The magnitude and duration of ISG and inflammatory gene expression was evaluated by gene expression profiling, where 5¡¯pppRNA triggered a sustained and diverse range of antiviral and inflammatory genes compared to treatment with IFN, and bioinformatics analysis identified distinct nodes of IRF7, IRF1 and NF-kB gene activation. Pre-treatment of A549 cells with 5¡¯pppRNA dramatically blocked H1N1 A/PR/8/34 Influenza virus replication; furthermore, intravenous delivery of 5¡äpppRNA to BALB/c mice generated an antiviral response in mouse lungs that protected animals from a lethal challenge with H1N1 A/PR/8/34. RNA agonist delivery inhibited virus replication in mouse lungs within the first 24h after H1N1 challenge and protected the lungs of infected animals from virus-induced pathology. Finally, 5¡¯pppRNA pre-treatment also completely or partially blocked replication of Dengue, Vaccinia and HIV-1 replication. These results illustrate that naturally derived RIG-I agonists represent a potent stimulator of the innate antiviral response, with the capacity to block replication of multiple pathogenic human viruses.
Termination of IFN signalling is likewise crucial to the proper maintenance of the innate and adaptive immune response to virus infection. We also identified an essential role for LUBAC-mediated linear ubiquitination of NEMO in the negative regulation of the RIG-I antiviral pathway through sequestration of TRAF3 from the MAVS adapter. LUBAC and NEMO-Ub constructs inhibited RIG-I signaling downstream of MAVS and upstream of TBK1; linearly ubiquitinated NEMO interacted physically with TRAF3, and disrupted the MAVS-TRAF3 complex, thus providing a mechanistic explanation for the downregulation of RIG-I signaling. Using SHARPIN deficient cpdm MEFs, we observed on the one hand, an increased and prolonged antiviral response, while on the other hand, an impaired NF-B activation, indicating that linear ubiquitination is required for NF-B activation downstream of RIG-I. Interestingly, an increase in apoptotic cell death was also detected in SHARPIN-deficient cpdm MEFs after VSV infection, potentially attributable to the absence of the anti-apoptotic activity of NF-B. These studies reveal a novel negative feedback mechanism used by host cells to regulate the IFN antiviral response.
