07-151 | Anti-PKR Antibody

The host antiviral protein kinase R (PKR) has rapidly evolved during primate evolution, likely in response to challenges posed by many different viral antagonists, such as the TRS1 gene of cytomegaloviruses (CMVs). In turn, viral antagonists have adapted to changes in PKR. As a result of this "arms race," modern TRS1 alleles in CMVs may function differently in cells derived from alternative species. We have previously shown that human CMV TRS1 (HuTRS1) blocks the PKR pathway and rescues replication of a vaccinia virus mutant lacking its major PKR antagonist in human cells. We now demonstrate that HuTRS1 does not have these activities in Old World monkey cells. Conversely, the rhesus cytomegalovirus homologue of HuTRS1 (RhTRS1) fulfills these functions in African green monkey cells, but not rhesus or human cells. Both TRS1 proteins bind to double-stranded RNA and, in the cell types in which they can rescue VVΔE3L replication, they also bind to PKR and prevent phosphorylation of the α-subunit of eukaryotic initiation factor 2. However, while HuTRS1 binds to inactive human PKR and prevents its autophosphorylation, RhTRS1 binds to phosphorylated African green monkey PKR. These studies reveal that evolutionary adaptations in this critical host defense protein have altered its binding interface in a way that has resulted in a qualitatively altered mechanism of PKR antagonism by viral TRS1 alleles from different CMVs. These results suggest that PKR antagonism is likely one of the factors that contributes to species specificity of cytomegalovirus replication.

The double-stranded (ds) RNA activated protein kinase PKR is an interferon (IFN)-inducible serine/threonine protein that regulates protein synthesis through the phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2alpha). PKR activation in cells is induced by virus infection or treatment with dsRNA and is modulated by a number of viral and cellular factors. To better understand the mechanisms of PKR action we have analyzed and compared the mode of PKR activation in a number of cell lines of different histological origin. Here we show that PKR activation and phosphorylation of eIF-2alpha are both diminished in various virus-transformed and nontransformed human T cells. Priming of T cells with IFN does not restore PKR activation. In vitro kinase assays show that the diminished PKR activation in T cells correlates with the presence of a 60-kDa (p60) phosphoprotein coimmunoprecipitated with PKR. P60 is absent from PKR immunoprecipitates from non T cells. Incubation of active PKR with T cell extracts results in inhibition of PKR autophosphorylation, which is proportional to the amount of phosphorylated p60 in the kinase reactions. Treatment of T cells with proteasome inhibitors or incubation of PKR immunoprecipitates with phosphatase inhibitors does not restore PKR activation. However, phosphorylation of p60 is enhanced upon treatment with the phosphatase inhibitor microcystin. These data show that the impaired activation capacity of PKR in human T cells is exerted at the post-translational levels in a manner that is independent of cell transformation or virus infection.

Protein kinase RNA-regulated (PKR) is an established component of innate antiviral immunity. Recently, PKR has been shown to be essential for signal transduction in other situations of cellular stress. The relationship between PKR and the stress-activated protein kinases (SAPKs), such as p38 mitogen-activated protein kinase (MAPK), is not clear. Using embryonic fibroblasts from PKR wild-type and null mice, we established a requirement for PKR in the activation of SAPKs by double-stranded RNA, lipopolysaccharide (LPS) and proinflammatory cytokines. This does not reflect a global failure to activate SAPKs in the PKR-null background as these kinases are activated normally by anisomycin and other physicochemical stress. Activation of p38 MAPK was restored in immortalized PKR-null cells by reconstitution with human PKR. We also show that LPS induction of interleukin-6 and interleukin-12 mRNA is defective in PKR-null cells, and that production of these cytokines is impaired in PKR-null mice challenged with LPS. Our findings indicate, for the first time, that PKR is required for p38 MAPK signaling and plays a potentially important role in the innate response against bacterial endotoxin.

The interferon-induced dsRNA-dependent protein kinase (PKR) induces apoptosis of mammalian cells. Apoptosis induction by PKR involves phosphorylation of the translational factor eIF-2alpha and activation of the transcriptional factor NF-kappaB, but caspase pathways activated by PKR are not known. Upregulation of Fas mRNA by PKR has been suggested to play a role in PKR-induced apoptosis. To learn how PKR induces apoptosis, we have analysed the role of molecules in death receptor pathways. We showed the involvement of the FADD-caspase 8 pathway on PKR-induced apoptosis based on four experimental findings: upregulation of caspase 8 activity during PKR-induced apoptosis, blocking of PKR-induced apoptosis by the use of a chemical inhibitor of caspase 8, and inhibition of PKR-induced apoptosis by expression of both a FADD dominant negative or a viral FLIP molecule. Significantly, despite the PKR-mediated upregulation of Fas mRNA expression, the Fas receptor-ligand pathway is not needed for PKR-induced apoptosis. Antibodies that inhibit TNFalpha-TNFR1 or Fas-FasL interactions were not able to block PKR-induced apoptosis. Taken together, our observations establish the involvement of caspase 8 in PKR-induced apoptosis and suggest that death receptors other than Fas or TNFR1 or, alternatively, a novel mechanism involving FADD independently of death receptors, are responsible for PKR-induced apoptosis.

Expression of the tumor suppressor IRF-1 results in the inhibition of cell growth and transcriptional activation of the IFN-beta gene. IFN production is not responsible for the IRF-1 mediated cell growth inhibition. It is shown here that activation of the IRF-1 causes induction of PKR expression. PKR is a serine/threonine kinase with tumor suppressor activity. IRF-1 mediated cell growth inhibition and IFN induction correlates with PKR expression. A catalytically inactive dominant negative PKR mutant abolishes both activities of IRF-1. These data demonstrate that the tumor suppressor activity of IRF-1 is mediated, at least in part, by PKR.