Roles for the double-stranded RNA-dependent protein kinase PKR in apoptosis and innate immunity to viral infection.

摘要

The interferons (IFNs) are a family of pleiotropic cytokines that exert their effects through the induction of more than thirty responsive genes. An important interferon-induced gene is the double-stranded (ds)RNA-dependent protein kinase PKR. This kinase inhibits translation of viral RNA following infection of the cell by phosphorylating the a subunit of eukaryotic translation initiation factor 2 (eIF2α) and inactivating it.; To further investigate the growth-suppressive and anti-viral properties of PKR, we established murine fibroblast cell lines that inducibly express either wild type PKR or a dominant negative PKR variant. We found that cells overexpressing wild type PKR underwent rapid Fas-associated death domain containing protein (FADD)-dependent apoptosis after activation of PKR by dsRNA treatment. Coincident with the induction of apoptosis, a PKR-dependent increase in the levels of the death receptor Fas [CD95/Apo-1] were observed. In contrast, cells expressing the dominant negative PKR variant became transformed and were completely resistant to apoptosis induced by dsRNA treatment or Fas ligation.; We also determined that cells over-expressing wild-type PKR were significantly more sensitive than control cells to apoptosis induced by influenza virus, but not by vesicular stomatitis virus (VSV) or Sindbis virus. Influenza virus-induced cell death was demonstrated to occur via the FADD/caspase 8 death signaling pathway, whereas VSV and Sindbis virus primarily triggered apoptosis through Apaf-1 mediated activation of caspase 9. While IFN pre-treatment greatly enhanced influenza virus- and dsRNA-induced cell death, it completely prevented apoptosis triggered by VSV and Sindbis virus.; Finally, we evaluated the role of PKR in innate immunity to viral infection by examining the susceptibility of mice lacking PKR to infection by selected RNA viruses. We found that PKR-deficient, but not wild type, mice were extremely susceptible to VSV and influenza virus infection. We further showed that PKR inhibits VSV replication at the level of viral mRNA translation by phosphorylating eIF2α, and that both PKR and autocrine IFN are required for the complete protection of primary wild type embryonic fibroblasts. The ability of PKR to encumber viral replication is thus non-redundant, and likely allows time for the IFN response and the adaptive immune system to completely eliminate the infectious agent.