Classical swine fever virus N^pro antagonises IRF3 to prevent IFN-independent TLR3 and RIG-I-mediated apoptosis

Classical swine fever virus (CSFV) is the causative agent of classical swine fever, a notifiable disease of economic importance that causes severe leukopenia, fever and haemorrhagic disease in domesticated pigs and wild boar across the globe. CSFV has been shown to antagonise the induction of type I IFN, partly through a function of its N-terminal protease (N^pro) which binds IRF3 and targets it for proteasomal degradation. Additionally, N^pro has been shown to antagonise apoptosis triggered by the dsRNA-homolog poly(I:C), however the exact mechanism by which this is achieved has not been fully elucidated. In this study we confirm the ability of N^pro to inhibit dsRNA-mediated apoptosis and show that N^pro is also able to antagonise Sendai virus-mediated apoptosis in PK-15 cells. Gene edited PK-15 cell lines were used to show the dsRNA-sensing pathogen recognition receptors (PRRs) TLR3 and RIG-I specifically respond to poly(I:C) and SeV respectively, subsequently triggering apoptosis through pathways that converge on IRF3 and culminate in the cleavage of caspase-3. Importantly, this IRF3-mediated apoptosis was found to be dependent on transcription-independent functions of IRF3 and also on Bax, a pro-apoptotic Bcl-2 family protein, through a direct interaction between the two proteins. Deletion of IRF3, stable expression of N^pro and infection with wild-type CSFV were found to antagonise the mitochondrial localisation of Bax, a key hallmark of the intrinsic, mitochondrial pathway of apoptosis. Together, these findings show that N^pro's putative interaction with IRF3 is involved not only in its antagonism of type I IFN, but also dsRNA-mediated mitochondrial apoptosis.Importance Responsible for severe haemorrhagic disease in domestic pigs and wild boar, classical swine fever is recognised by the World Organisation for Animal Health (OIE) and European Union as a notifiable disease of economic importance. Persistent infection, immunotolerance and early dissemination of the virus at local sites of infection have been linked to the antagonism of type I IFN induction by N^pro This protein may further contribute to these phenomena by antagonising the induction of dsRNA-mediated apoptosis. Ultimately, apoptosis is an important innate mechanism by which cells counter viruses at local sites of infection, thus preventing wider spread and dissemination within the host, potentially also contributing to the onset of persistence. Elucidation of the mechanism by which Npro antagonises the apoptotic response will help inform the development of rationally-designed live-attenuated vaccines and antivirals for control of outbreaks in typically CSFV-free countries.