Classical Swine Fever Virus Npro Antagonizes IRF3 To Prevent Interferon-Independent TLR3- and RIG-I-Mediated Apoptosis

Responsible for severe hemorrhagic disease in domestic pigs and wild boar, classical swine fever is recognized 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 Npro. ABSTRACT 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 hemorrhagic disease in domesticated pigs and wild boar across the globe. CSFV has been shown to antagonize the induction of type I interferon (IFN), partly through a function of its N-terminal protease (Npro), which binds interferon regulatory factor 3 (IRF3) and targets it for proteasomal degradation. Additionally, Npro has been shown to antagonize apoptosis triggered by the double-stranded RNA (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 Npro to inhibit dsRNA-mediated apoptosis and show that Npro is also able to antagonize Sendai virus (SeV)-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 proapoptotic Bcl-2 family protein, through a direct interaction between the two proteins. Deletion of IRF3, stable expression of Npro, and infection with wild-type CSFV were found to antagonize the mitochondrial localization of Bax, a key hallmark of the intrinsic, mitochondrial pathway of apoptosis. Together, these findings show that Npro’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 hemorrhagic disease in domestic pigs and wild boar, classical swine fever is recognized 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 Npro. This protein may further contribute to these phenomena by antagonizing 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 antagonizes 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.