The role of PKR and eIF2alpha in defense against infection by infectious pancreatic necrosis virus in rainbow trout

Abstract

The role of eIF2alpha and PKR in the antiviral activity of interferon has been well characterized in mammals. Although an interferon response has been described in fish, response to virus infection at the level of eIF2alpha phosphorylation has not been demonstrated. The cDNA for eIF2alpha, the substrate of PKR, has been cloned and sequenced from zebrafish (Danio rerio ) and rainbow trout. The deduced amino acid sequences of zebrafish and rainbow trout eIF2alpha are 93% and 91% identical to the human protein, respectively, and regions corresponding to both the phosphorylation site of eIF2alpha and the kinase-docking site are identical in both fish to the human amino acid sequence. The transcripts of both fish cDNAs encode proteins with similar characteristics to human eIF2alpha. Both proteins can be phosphorylated in vitro and in vivo by several eIF2alpha-kinases. Furthermore, an eIF2alpha-kinase has been isolated by affinity-chromatography from the RTG-2 rainbow trout cell line. Based on several characteristics, this kinase has been identified as rainbow trout PKR (rtPKR). As well as being able to bind dsRNA, rtPKR activity is stimulated by a Type-1 interferon-like activity and demonstrates a biphasic response to increasing concentrations of dsRNA. The putative rtPKR is able to phosphorylate recombinant wild-type human eIF2alpha in vitro, but not the Ser51Ala variant of eIF2alpha that lacks the PKR phosphorylation site. rtPKR also phosphorylates the newly identified substrate, NFAR2. Rainbow trout eIF2alpha becomes phosphorylated in interferon treated RTG-2 cells in response to infectious pancreatic necrosis virus (IPNV), with phosphorylation increasing with severity of infection. Additionally, inhibition of rtPKR activation with 2-aminopurine prevents eIF2alpha phosphorylation and increases viral pathogenesis. The most prominent finding is that a Type-I interferon-like activity confers a protective effect in salmonids through activation of PKR and increased eIF2alpha phosphorylation, as in mammals. This study establishes a link between the ability of interferon to protect RTG-2 cells from IPNV infection and the increased phosphorylation of eIF2alpha by PKR. These data represent that first direct evidence of the efficacy of the interferon/PKR/eIF2alpha host defense pathway in organisms lower than mammals and demonstrate that this pathway may be an ancient vertebrate protective mechanism.