| Type I interferons (IFNs) are critical mediators of host defense that rapidly induce an antiviral response through transcriptional regulation of target genes. IFN signaling is induced when pathogens are detected by cellular pattern recognition receptors. As a result, viruses have evolved multiple antagonists to target IFN signaling.;Vaccinia Virus (VV) is a large double-stranded DNA (dsDNA) virus, which is known to antagonize both innate and adaptive immune responses. VV E3L gene product has previously been shown to target IFN effectors such as the dsRNA-dependent protein kinase (PKR) and 2'-5'oligoadenylate synthase (OAS). However, previous work in our lab had suggested a novel role for E3L as an inhibitor of IFN induction. Therefore, we sought to characterize the molecular ligand of VV responsible for triggering IFN induction, as well as the molecular pathways that mediate IFN induction in response to VV infection, in order to identify E3L targets, and to uncover the mechanism(s) by which it impairs IFN signaling.;We found that IFN induction is dramatically enhanced in the absence of E3L, and that this phenotype requires expression of both tank binding kinase 1 (TBK1) and I-kappa-B-kinase epsilon (IKK-epsilon). We also observed that IFN induction during VV infection requires the IFN-beta-promoter stimulator 1(IPS-1), and identified the melanoma differentiation-associated gene 5 protein (MDA-5) as the key PRR that is activated in response to VV. Interestingly, we also showed that the IFN induction is dependent on the expression of PKR, but that its canonical target is not involved.;Using co-immunoprecipitation we found that E3L associates with MDA-5 and IKK-epsilon; in a dsRNA- and phosphorylation-dependent manner, and that E3L is phosphorylated on specific residues in the presence of IKK-epsilon;. Furthermore, we demonstrated that inhibition of E3L targets is a crucial step in VV pathogenesis by showing that VV replication is enhanced in MDA-5- and IKK-epsilon-deficient cells.;Taken together, our data suggest that VV stimulates a cytoplasmic signaling cascade involving MDA-5, MAVS, TBK1, IKK-epsilon, and PKR, leading to type I IFN induction. However, E3L mounts a multi-pronged attack on the host IFN response by inhibiting these cascades via direct protein-protein interaction with critical signaling factors. |
