In this work, we report cDNA cloning of two type I interferons (IFNs) from the head kidney of Atlantic salmon, called SasaIFN-alpha1 (829 bp) and SasaIFN-alpha2 (1290 bp). Both translate into 175 amino acid precursor molecules showing 95% amino acid sequence identity. The precursors have a putative 23 amino acid signal peptide, which suggests that the mature Atlantic salmon IFNs contain 152 amino acids (18.2 kDa). Salmon IFN appears to have five alpha-helices, similar to mammalian and avian type I IFNs, and showed 45% sequence identity with zebrafish IFN, up to 29% identity with mammalian IFN-alpha sequences, and 17%-18% sequence identity with mammalian IFN-beta and chicken type I IFNs. Human embryonic kidney 293 cells transfected with the SasaIFN-alpha1 cDNA gene produced high titers of acid-stable antiviral activity, which protected salmonid cells against infectious pancreatic necrosis virus (IPNV) and also induced Mx protein in the cells. Poly(I)-poly(C) induced two IFN transcripts in head kidney of Atlantic salmon. Genomic IFN sequences contained four introns and five exons, which is different from the intronless type I IFN genes of birds and mammals.

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A common strategy in the cellular response to stress signals is to shut down protein synthesis [1]. The cell contains several different latent translation initiation factor 2 alpha (eIF2a)-kinases that phosphorylate the a-subunit of the translation initiation factor eIF2 upon certain stimuli, such as viral infection, amino acid starvation, heme-depletion, and accumulation of misfolded proteins in the endoplasmic reticulum, leading to inhibition of protein synthesis [2]. Under normal conditions, eIF2 associates with GTP and Met-tRNA i to form a complex with the 40S ribosomal subunit [3]. As initiation proceeds, eIF2 responds to alignment of the initiator tRNA with the initiation codon by initiating hydrolysis of GTP and releasing Met-tRNA i into the partial P-site. Recycling of eIF2-GDP to the active GTP-bound form requires the GTP exchange factor, The translation initiation factor 2 alpha (eIF2a)-kinase, dsRNA-activated protein kinase (PKR), constitutes one of the major antiviral proteins activated by viral infection of vertebrates. PKR is activated by viral double-stranded RNA and subsequently phosphorylates the a-subunit of translation initiation factor eIF2. This results in overall down regulation of protein synthesis in the cell and inhibition of viral replication. Fish appear to have a PKR-like protein that has Z-DNA binding domains instead of dsRNA binding domains in the regulatory domain, and has thus been termed Z-DNA binding protein kinase (PKZ). We present the cloning of the Atlantic salmon PKZ cDNA and show its upregulation by interferon in Atlantic salmon TO cells and poly inosinic poly cytodylic acid in head kidney. We also demonstrate that recombinant Atlantic salmon PKZ, expressed in Escherichia coli, phosphorylates eIF2a in vitro. This is the first demonstration that PKZ is able to phosphorylate eIF2a. PKZ activity, as measured by phosphorylation of eIF2a, was increased after addition of Z-DNA, but not by dsRNA. In addition, we show that wild-type Atlantic salmon PKZ, but not the kinase defective variant K217R, has a direct inhibitory effect on protein synthesis after transient expression in Chinook salmon embryo cells. Overall, the results support a role for PKZ, like PKR, in host defense against virus infection.Abbreviations As, Atlantic salmon; As-IFN, Atlantic salmon interferon; b-gal, b-galactosidase; CHSE, Chinook salmon embryonic; dsRBD, dsRNA binding domain; EGFP, enhanced green fluorescence protein; eIF2a, translation initiation factor 2 alpha; EST, expressed sequence tag; Hu, human; IFN, interferon; PERK, protein kinase-like endoplasmic reticulum eIF2a kinase; PKR, dsRNA-activated protein kinase; PKZ, Z-DNA binding protein kinase; poly dG:dC, poly deoxyguanosine poly deoxycytidine acid; poly I:C, poly inosinic poly cytodylic acid; SG, stress granule; ZBP1, Z-DNA binding protein 1.

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The type I interferon (IFN) system plays a critical role in the innate immune defense against viruses in vertebrates. Virus-infected cells synthesize and secrete type I interferons (IFN-a ⁄ b), which circulate in the body and protect other cells from viral infection. The antiviral action is caused by binding of IFN-a ⁄ b to the type I IFN receptor resulting in activation of transcription of several hundred IFN-stimulated genes, some of which encode proteins that inhibit viral replication. The antiviral properties of at least three type I IFNinduced proteins are well established. These comprise dsRNA-activated protein kinase R (PKR), 2¢,5¢-oligoadenylate synthetase and Mx proteins [1].Although the structures of the IFN-a and IFN-b promoters from human and mouse have been long known, the mechanisms involved in viral induction of type I IFNs have only recently been uncovered [2,3]. Of great importance has been the discovery of IFN super-producing blood cells called plasmacytoid dendritic cells (pDCs) and the realization that the Keywords Atlantic salmon; interferon promoter; interferon regulatory factor; nuclear factor kappa B (NFjB); poly(I:C) Correspondence B. Robertsen,