| The interferon-inducible, RNA-dependent protein kinase PKR is an important effector of antiviral and antiproliferative activities of interferons. Although the best characterized function of PKR is the inhibition of translation, more recently PKR also has been shown to modulate signal transduction processes and regulate gene expression. Upon RNA binding, PKR is autophosphorylated and then subsequently catalyzes the phosphorylation of protein synthesis initiation factor eIF2alpha, the best known cellular substrate of PKR. Here we analyzed the function of PKR in translational control as well as in activation of IFN-beta gene expression in response to measles virus infection. A complementation assay strategy was carried out by expressing PKR in PKR-deficient (PKR kd) human cells stably knocked down for PKR protein expression by a shRNA silencing approach. As a comparative analysis, the function of the Z-DNA binding protein PKZ, which has a kinase domain similar to that of PKR, was also assessed by PKZ expression in human PKRkd cells. We first demonstrated that in the complemented PKRkd cells in the absence of infection, PKR as well as PKZ both mediated the inhibition of cap-dependent translation in a manner that depended upon catalytic activity. In contrast, IRES-dependent translation was not affected. While PKR-mediated inhibition of reporter expression was correlated with an elevated level of eIF2alpha phosphorylation at serine 51, no detectable increase in phosphorylated eIF2alpha was observed in PKZ expressing cells. Furthermore, C-deficient (Cko) measles virus infection of complemented PKRkd cells revealed that both RNA-binding and catalytic activities of PKR are required for PKR-dependent activation of the IFN-beta signaling pathway and induction of IFN-beta gene. Neither a catalytic mutant form of PKR (K296R), nor the related kinase PKZ were able to mediate activation of ATF2, JNK and p38, or enhance the induction of IFN-beta gene; and only wildtype PKR but not the K296R or PKZ reduced the growth of Cko virus in PKRkd cells. In addition, PKR enhanced the IFN-beta transcript levels through translational control by phosphorylation of translation initiation factor eIF2alpha. The expression of a phosphorylation defective form of eIF2alpha (S48,51A) in PKR-sufficient cells reduced the induction of IFN-beta and enhanced the production of IkappaB-alpha, the inhibitor of the IFN-beta transcription factor, NF-kappaB, following Cko virus infection. In conclusion, these studies reveal the importance of translational control function of PKR through phosphorylation of eIF2alpha in suppression of gene expression and induction of IFN gene following virus infection or aberrant RNA generation during transfection. |
