Classical Swine Fever Virus N^proAntagonizes IRF3 To Prevent Interferon-Independent TLR3- and RIG-I-Mediated Apoptosis

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 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 (Npro) which binds IRF3 and targets it for proteasomal degradation. Additionally, Npro 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 Npro to inhibit dsRNA-mediated apoptosis and show that Npro 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 Npro 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 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 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 Npro. 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.

Analysis of porcine bone marrow-derived macrophage cytokine responses to infection with PRRSV-1 strains of differing virulence

“…Having observed strain dependent differences in the expression of IFN-a, we thought it necessary to investigate the type I IFN response in PRRSV-1 infected BMDM. We have previously demonstrated that dsRNA and SeV are specific agonists of TLR-3 and RIG-I mediated type I IFN responses in porcine cells, respectively (37). When utilized in BMDM, both ligands induced strong expression of the ISGs, Mx1, ISG15 and RIG-I.…”

“…Western blots were performed as previously described (37). Primary antibodies; Anti-RIG-I (Santa Cruz, Dallas, US #sc-376845), Anti-Mx1 (GeneTex, Hsinchu City 300 Taiwan, R.O.C., #GTX110256), Anti-ISG15 (Abcam, Cambridge, UK, #ab233071), Anti-g-tubulin (Merck, #T6557) were used where indicated.…”

“…Having observed that infection with virulent SU1-Bel induced significantly higher expression of IFN-a compared to the less virulent 215-06 and Olot/91 strains, we postulated that this may lead to a higher expression of type I IFN stimulated genes (ISG). We have previously validated the expression of Mx1, RIG-I and ISG15 as markers of ISG expression in immortalized porcine cells (37) and applied this methodology to investigate ISG expression in the porcine BMDM. To facilitate this, BMDM from three animals were infected (MOI 0.01) with either SU1-Bel, 215-06 or Olot/91 and at 48 hpi whole cell lysates were prepared and analyzed for expression of Mx1, RIG-I and ISG15 by Western blot (Figure 4).…”

IRF3 inhibits nuclear translocation of NF-κB to prevent viral inflammation

“…Viruses often evade the immune response by antagonizing specific pathways. Numerous viral proteins have been shown to antagonize the transcriptional and the RIPA activity of IRF3 ( 80 – 82 ). It is also tempting to speculate the viruses have coevolved to evade specific functions of IRF3, and the host can fight back using the additional options.…”

Structure and Function of Mitochondria‐Associated Endoplasmic Reticulum Membranes (MAMs) and Their Role in Cardiovascular Diseases

“…In addition, STING is a target for HCV-NS4B to block RIG-I-mediated activation of IFN-β production by blocking its interaction with MAVS. NS4B and STING are localized on ER and MAM [139]. MAM-resident factor Gp78 was also found to be involved in innate antiviral signaling by targeting MAVS during vesicular stomatitis virus (VSV) infection [140].…”