Abstract
The sera from pigs infected with virulent classical swine fever virus (CSFV) contain substantial amounts of tumor necrosis factor (TNF), a prototype proinflammatory cytokine with pleiotropic activities. TNF limits the replication of CSFV in cell culture. In order to investigate the signaling involved in the antiviral activity of TNF, we employed small-molecule inhibitors to interfere specifically with JAK/STAT and NF-κB signaling pathways in near-to-primary endothelial PEDSV.15 cells. In addition, we knocked out selected factors of the interferon (IFN) induction and signaling pathways using CRISPR/Cas9. We found that the anti-CSFV effect of TNF was sensitive to JAK/STAT inhibitors, suggesting that TNF induces IFN signaling. Accordingly, we observed that the antiviral effect of TNF was dependent on intact type I IFN signaling as PEDSV.15 cells with the disrupted type I IFN receptor lost their capacity to limit the replication of CSFV after TNF treatment. Consequently, we examined whether TNF activates the type I IFN induction pathway. With genetically modified PEDSV.15 cells deficient in functional interferon regulatory factor 1 or 3 (IRF1 or IRF3), we observed that the anti-CSFV activity exhibited by TNF was dependent on IRF1, whereas IRF3 was dispensable. This was distinct from the lipopolysaccharide (LPS)-driven antiviral effect that relied on both IRF1 and IRF3. In agreement with the requirement of IRF1 to induce TNF- and LPS-mediated antiviral effects, intact IRF1 was also essential for TNF- and LPS-mediated induction of IFN-β mRNA, while the activation of NF-κB was not dependent on IRF1. Nevertheless, NF-κB activation was essential for the TNF-mediated antiviral effect. Finally, we observed that CSFV failed to counteract the TNF-mediated induction of the IFN-β mRNA in PEDSV.15 cells, suggesting that CSFV does not interfere with IRF1-dependent signaling. In summary, we report that the proinflammatory cytokine TNF limits the replication of CSFV in PEDSV.15 cells by specific induction of an IRF1-dependent antiviral type I IFN response.
Highlights
The PEDSV.15 cells stimulated for six hours with increasing concentrations of murine TNF (mTNF), from 0.4 ng/mL to
In the PEDSV.15 cells, we demonstrated that the tumor necrosis factor (TNF)-triggered induction of IFN-β transcripts and the resulting antiviral effect rely on intact IRF1 (Figure 4b,d), whereas IRF3 was dispensable (Figure 3d)
The anti-classical swine fever virus (CSFV) activity of porcine and murine TNF was inhibited by antibody-mediated TNF neutralization, nuclear factor κB (NF-κB)
Summary
The first line of protection of host cells from invading viruses is mediated by the innate immune system. By sensing unique pathogen-associated molecular patterns, conserved cellular pattern recognition receptors initiate multiple intracellular signaling cascades involving interferon regulatory factors (IRF) that culminate in the transcriptional activation and secretion of type I interferons (IFN-α and IFN-β) and type III IFN (IFN-λ) [1]. Specific interactions of IFNs with cellular type I (IFNAR1) and type III IFN receptors subsequently activate Janus kinase (JAK)- and signal transducer and activator of transcription (STAT)dependent signaling in neighboring cells. The IFN-mediated JAK/STAT signaling leads to the expression of a multitude of IFN-stimulated genes that synergistically orchestrate cellular antiviral defense [2]
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