The double-stranded RNA-dependent protein kinase (PKR) is one of the four mammalian kinases that phosphorylates the translation initiation factor 2␣ in response to virus infection. This kinase is induced by interferon and activated by double-stranded RNA (dsRNA). Phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) blocks translation initiation of both cellular and viral mRNA, inhibiting virus replication. To counteract this effect, most viruses express inhibitors that prevent PKR activation in infected cells. Here we report that PKR is highly activated following infection with alphaviruses Sindbis (SV) and Semliki Forest virus (SFV), leading to the almost complete phosphorylation of eIF2␣. Notably, subgenomic SV 26S mRNA is translated efficiently in the presence of phosphorylated eIF2␣. This modification of eIF2 does not restrict viral replication; SV 26S mRNA initiates translation with canonical methionine in the presence of high levels of phosphorylated eIF2␣. Genetic and biochemical data showed a highly stable RNA hairpin loop located downstream of the AUG initiator codon that is necessary to provide translational resistance to eIF2␣ phosphorylation. This structure can stall the ribosomes on the correct site to initiate translation of SV 26S mRNA, thus bypassing the requirement for a functional eIF2. Our findings show the existence of an alternative way to locate the ribosomes on the initiation codon of mRNA that is exploited by a family of viruses to counteract the antiviral effect of PKR.[Keywords: Translation; eIF2; eIF2A; PKR; alphaviruses; antiviral response] Supplemental material is available at http://www.genesdev.org.
These results provide a mechanism for translation inhibition by UV irradiation and identify a hitherto unrecognized role for mammalian GCN2 as a mediator of the cellular response to UV stress.
In eukaryotic cells, protein synthesis is regulated in response to various environmental stresses by phosphorylating the a subunit of the eukaryotic initiation factor 2 (eIF2a). Three different eIF2a kinases have been identified in mammalian cells, the heme-regulated inhibitor (HRI), the interferon-inducible RNA-dependent kinase (PKR) and the endoplasmic reticulum-resident kinase (PERK). A fourth eIF2a kinase, termed GCN2, was previously characterized from Saccharomyces cerevisiae, Drosophila melanogaster and Neurospora crassa. Here we describe the cloning of a mouse GCN2 cDNA (MGCN2), which represents the first mammalian GCN2 homolog. MGCN2 has a conserved motif, N-terminal to the kinase subdomain V, and a large insert of 139 amino acids located between subdomains IV and V that are characteristic of the known eIF2a kinases. Furthermore, MGCN2 contains a class II aminoacyl-tRNA synthetase domain and a degenerate kinase segment, downstream and upstream of the eIF2a kinase domain, respectively, and both are singular features of GCN2 protein kinases. MGCN2 mRNA is expressed as a single message of < 5.5 kb in a wide range of different tissues, with the highest levels in the liver and the brain. Specific polyclonal anti-(MGCN2) immunoprecipitated an eIF2a kinase activity and recognized a 190 kDa phosphoprotein in Western blots from either mouse liver or MGCN2-transfected 293 cell extracts. Interestingly, serum starvation increased eIF2a phosphorylation in MGCN2-transfected human 293T cells. This finding provides evidence that GCN2 is the unique eIF2a kinase present in all eukaryotes from yeast to mammals and underscores the role of MGCN2 kinase in translational control and its potential physiological significance.
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