Protein kinase PKR amplification of interferon ß induction occurs through initiation factor eIF-2α-mediated translational control.

The protein kinase PKR is activated by RNA with double-stranded (ds) structure and subsequently impairs translation through phosphorylation of protein synthesis initiation factor eIF-2α. PKR also mediates activation of signal transduction pathways leading to interferon beta (IFN-ß) gene induction following virus-infection or RNA transfection. We previously demonstrated in measles virus-infected cells that PKR is required for the maximal induction of IFN-ß gene expression by the interferon promoter stimulator gene 1 (IPS-1) adaptor-dependent cytosolic RNA sensor pathway. While both IPS-1 and PKR are important mediators of IFN-ß induction, with PKR contributing to an enhanced NF-κB activation, the mechanism by which PKR enhances NF-κB activity and amplifies IFN-ß induction is unresolved. Herein we tested the possibility that PKR could activate signal transduction pathways indirectly through translational control responses. Following transfection with synthetic or natural dsRNAs or infection with measles virus, we observed increased mRNA but decreased protein levels for the inhibitor of NF-κB signaling, IκB-α, that correlated with PKR activation and eIF-2α phosphorylation. Importantly, knockdown of PKR increased IκB-α protein levels and impaired IFN-ß induction. Additionally, inhibition of translation by cycloheximide treatment rescued IFN-ß induction following PKR knockdown but not IPS-1 knockdown. Mutation of eIF-2α to prevent phosphorylation also impaired IFN-ß induction in PKR-sufficient virus-infected cells. These results suggest that an eIF-2α-dependent translation inhibition mechanism is sufficient to explain the PKR-mediated amplification of IPS-1-dependent IFN-ß induction by foreign RNA.

RNA-dependent protein kinase PKR and the Z-DNA binding orthologue PKZ differ in their capacity to mediate initiation factor eIF2α-dependent inhibition of protein synthesis and virus-induced stress granule formation.

Protein kinase R (PKR), a regulator of translation in mammalian cells, possesses two ds-RNA binding domains responsible for kinase activation. Protein kinase Z (PKZ), a PKR-like kinase present in fish, possesses two Z-DNA binding domains. A complementation strategy with cells stably deficient in PKR was used to compare the functions of PKR and PKZ. We found reporter expression was inhibited by wildtype (WT) PKR but not by either catalytic (K296R) or RNA-binding (K64E) mutants. PKZ, like PKR, more potently inhibited 5' cap-dependent compared to IRES-dependent reporter expression. However, in contrast to PKR-expressing cells, phosphorylation of initiation factor eIF2α was not detectably increased in PKZ-expressing cells. Furthermore, virus-induced stress granule formation was observed in PKR-deficient cells complemented with WT PKR but not K296R mutant PKR or WT PKZ. These results suggest that PKR and PKZ function by distinguishable mechanisms to modulate host responses including protein synthesis inhibition and stress granule formation.

Protein kinase PKR catalytic activity is required for the PKR-dependent activation of mitogen-activated protein kinases and amplification of interferon beta induction following virus infection.

The protein kinase regulated by RNA (PKR) enhances both activation of mitogen-activated protein kinases and the induction of interferon beta (IFN-ß) by measles virus defective in C-protein expression (C(ko)). Here we used complementation of human cell lines stably deficient in PKR (PKR(kd)) to probe the basis of these PKR-mediated responses. We found that PKR(kd) HeLa and amnion U cell lines were defective for virus-mediated activation of IFN induction signaling components compared to PKR-sufficient control cells. Complementation of PKR(kd) cells with wildtype PKR, but not with PKR mutants defective in either catalytic activity or dsRNA-binding activity, restored JNK, p38 and ATF-2 phosphorylation and enhanced IFN-ß induction following infection. By contrast to mammalian PKR, the Z-DNA binding domain-containing fish homologue of PKR, PKZ, lacked the capacity to enhance C(ko) virus-mediated IFN-ß induction. Furthermore, inhibition of virus growth was observed with C(ko)-infected PKR(kd) cells complemented with PKR but not with PKZ.