Induction of Protein Kinase PKR-dependent Activation of Interferon Regulatory Factor 3 by Vaccinia Virus Occurs through Adapter IPS-1 Signaling

Interferon regulatory factor 3 (IRF-3) undergoes phosphorylation-induced activation in virus-infected cells and plays an important role in the antiviral innate immune response. The E3L protein encoded by vaccinia virus is known to impair phosphorylation and activation of IRF-3. Kinases in addition to I kappa B kinase-related kinases are implicated in the IRF-3-dependent antiviral response. To test in human cells the role of the protein kinase regulated by RNA (PKR) in IRF-3 activation, HeLa cells made stably deficient in PKR using an RNA interference strategy were compared with PKR-sufficient cells. Rapid phosphorylation and nuclear accumulation of IRF-3 were detected in PKR-sufficient cells following infection with E3L deletion mutant (Delta E3L) virus. By contrast, the full IRF-3 activation response was largely abolished in PKR-deficient cells. The Delta E3L virus-induced IRF-3 activation seen in PKR-sufficient cells was diminished by treatment with cytosine beta-D-arabinofuranoside. Furthermore, the vaccinia mutant ts23, which displays increased viral double-stranded RNA production at 39 C, induced PKR-dependent IRF-3 phosphorylation at 39 C but not at 31 C. Both IRF-3 phosphorylation and cell apoptosis induced by infection with Delta E3L virus were dependent upon RIG-I-like receptor signal transduction components, including the adapter IPS-1. These data suggest that PKR facilitates the host innate immune response and apoptosis in virus-infected cells by mediating IRF-3 activation through the mitochondrial IPS-1 signal transduction pathway.