IRF3 and IRF7 phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or I kappa B kinase but is blocked by vaccinia virus E3L protein

Induction of interferon-alpha (IFN alpha) gene expression in virus-infected cells requires phosphorylation-induced activation of the transcription fac tors IRF3 and IRF7. However, the kinase(s) that targets these proteins has not been identified. Using a combined pharmacological and genetic approach, we found that none of the kinases tested was responsible for IRF phosphory lation in cells infected with Newcastle disease virus (NDV). Although the b road spectrum kinase inhibitor staurosporine potently blocked IRF3 and -7 p hosphorylation, inhibitors for protein kinase C, protein kinase A, MEK, SAP K, IKK, and protein kinase R (PKR) were without effect. Both I kappaB kinas e and PKR have been implicated in IFN induction, but cells genetically defi cient in I kappaB kinase, PKR, or the PKR-related genes PERK; IRE1, or GCN2 retained the ability to phosphorylate IRF7 and induce IFN alpha. Interesti ngly, PKR mutant cells were defective for response to double-stranded (ds) RNA but not to virus infection, suggesting that dsRNA is not the only activ ating viral component. Consistent with this notion, protein synthesis was r equired for IRF7 phosphorylation in virus-infected cells, and the kinetics of phosphorylation and viral protein production were similar. Despite evide nce for a lack of involvement of dsRNA and PKR, vaccinia virus E3L protein, a dsRNA-binding protein capable of inhibiting PKR, was an effective IRF3 a nd -7 phosphorylation inhibitor. These results suggest that a novel cellula r protein that is activated by viral products in addition to dsRNA and is s ensitive to E3L inhibition is responsible for IRF activation and reveal a n ovel mechanism for the anti-IFN effect of E3L distinct from its inhibition of PKR.