Structural and functional studies of an IRF-7-like gene from Atlantic salmon.

Interferon regulatory factor 7 (IRF-7) plays a crucial role in virus-induced activation of interferon-alpha/beta transcription in mammals. This work describes a structural and functional homologue of mammalian IRF-7 from Atlantic salmon. The cloned gene encodes a putative protein of 415 amino acids (aa), which groups with mammalian IRF-7 and other fish IRF-7-like proteins in a phylogenetic analysis of vertebrate IRFs. Using an IFN promoter-luciferase assay we showed that salmon IRF-7 gave increased promoter activity after poly I:C stimulation. Transcript levels of IRF-7 were measured by real-time RT-PCR and compared to those of signal transducer and activator of transcription 1 (STAT1), which is important for transcriptional activation of IFN stimulated genes. Recombinant salmon IFN-alpha1 and poly I:C proved to be potent inducers of IRF-7 in Atlantic salmon TO cells, and poly I:C also induced the gene in head kidney and liver of Atlantic salmon. STAT1 was also induced by IFN, but was only weakly induced by poly I:C stimulation in vitro. Differences in transcription kinetics between IRF-7 and STAT1 thus indicate that the genes are regulated through different pathways. Finally, infection of TO cells with infectious salmon anemia virus (ISAV) induced early synthesis of STAT1 mRNA, whereas IRF-7 transcripts were upregulated much later. This indicates that ISAV has mechanisms to antagonize IRF-7 transcription and thus also the IFN system in Atlantic salmon.

The Atlantic salmon Z-DNA binding protein kinase phosphorylates translation initiation factor 2 alpha and constitutes a unique orthologue to the mammalian dsRNA-activated protein kinase R.

The translation initiation factor 2 alpha (eIF2alpha)-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 alpha-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 eIF2alphain vitro. This is the first demonstration that PKZ is able to phosphorylate eIF2alpha. PKZ activity, as measured by phosphorylation of eIF2alpha, 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.

Promoters of type I interferon genes from Atlantic salmon contain two main regulatory regions.

Recognition of viral nucleic acids by vertebrate host cells results in the synthesis and secretion of type I interferons (IFN-alpha/beta), which induce an antiviral state in neighboring cells. We have cloned the genes and promoters of two type I IFNs from Atlantic salmon. Both genes have the potential to encode IFN transcripts with either a short or a long 5'-untranslated region, apparently controlled by two distinct promoter regions, PR-I and PR-II, respectively. PR-I is located within 116 nucleotides upstream of the short transcript and contains a TATA-box, two interferon regulatory factor (IRF)-binding motifs, and a putative nuclear factor kappa B (NFkappaB)-binding motif. PR-II is located 469-677 nucleotides upstream of the short transcript and contains three or four IRF-binding motifs and a putative ATF-2/c-Jun element. Complete and truncated versions of the promoters were cloned in front of a luciferase reporter gene and analyzed for promoter activity in salmonid cells. Constructs containing PR-I were highly induced after treatment with the dsRNA poly(I:C), and promoter activity appeared to be dependent on NFkappaB. In contrast, constructs containing exclusively PR-II showed poor poly(I:C)-inducible activity. PR-I is thus the main control region for IFN-alpha/beta synthesis in salmon. Two pathogenic RNA viruses, infectious pancreatic necrosis virus and infectious salmon anemia virus, were tested for their ability to stimulate the minimal PR-I, but only the latter was able to induce promoter activity. The established IFN promoter-luciferase assay will be useful in studies of host-virus interactions in Atlantic salmon, as many viruses are known to encode proteins that prevent IFN synthesis by inhibition of promoter activation.

Atlantic salmon interferon genes: cloning, sequence analysis, expression, and biological activity.

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.

Characterization of Atlantic halibut (Hippoglossus hippoglossus) Mx protein expression.

Mx proteins are antiviral GTPases that are induced by type I interferons in vertebrates. An Atlantic halibut Mx cDNA (HHMx) was recently cloned. In this work, a polyclonal antiserum against HHMx protein was generated that detected a 71 kDa protein in the nuclei of Chinook salmon embryo cells transfected with the HHMx cDNA. Mx protein expression in organs of halibut was studied by immunoblot analysis after injection with the double-stranded RNA poly I:C or infectious pancreatic necrosis virus. Poly I:C stimulated increased Mx protein expression in liver, kidney, heart, spleen, gills and intestine. The Mx protein level in liver reached a maximum after 3 days and remained elevated for 14 days after treatment. IPNV infection resulted in increased Mx protein in liver from 4 to at least 35 days. Immunocytochemical detection of Mx proteins in blood smears from poly I:C treated halibut indicated that a cytoplasmic Mx form might exist in this species. Detection of Mx proteins in blood leukocytes could thus work as an early non-lethal test for viral infections.

Regulation and function of interferon regulatory factors of Atlantic salmon.

Transcription factors of the interferon regulatory factor (IRF) family are major regulators of the early immune responses against viral infections. In particular, IRF1, IRF2, IRF3 and IRF7 of mammals are known to regulate the expression of type I interferons (IFNs), which constitute the obligate cytokines for antiviral defense. We therefore cloned the coding sequence of Atlantic salmon (As) IRF1, IRF2, IRF3 and IRF7B. Expression profiles were studied in Atlantic salmon TO cells after poly I:C (dsRNA) transfection, treatment with recombinant salmon IFNa1 and infection with infectious salmon anemia virus (ISAV). The main findings were that AsIRF1 was earliest up-regulated by all stimuli, while AsIRF3 and AsIRF7 had a similar activation profile induced at a slightly later time point. The ability to induce the Atlantic salmon IFNa1 promoter was measured in a luciferase reporter assay. The results showed that AsIRF1, AsIRF3 and AsIRF7B were able to induce the promoter in a dose-dependent manner. AsIRF2 repressed the promoter, while AsIRF7A and a splicing variant (AsIRF3D) lacking the interaction domain had almost no effect. Combination of AsIRF1 and AsIRF3 had a synergistic stimulatory effect on the promoter compared to each of the two IRFs alone. Overall, our findings suggest that AsIRF3 is the main regulator of salmon IFNa1 production along with AsIRF1, which is less potent. This confirms a similar role for salmon IRF3 as mammalian IRF3 to be one of the main IRFs eliciting salmon IFNa1 production. Surprisingly, AsIRF7A and AsIRF7B seemed to have a lesser role in salmon IFNa1 induction, which may indicate that these factors have a larger role in activating other IFN genes or interferon stimulatory genes of Atlantic salmon.

Atlantic salmon IPS-1 mediates induction of IFNa1 and activation of NF-kappaB and localizes to mitochondria.

The striking difference in evolution of type I IFN genes of fish and mammals poses the question of whether these genes are induced through similar or different signalling pathways in the two vertebrate groups. Previous work has shown that expression of both Atlantic salmon (Salmo salar) IFNa1 and mammalian IFN-beta genes is dependent on IRF and NF-kappaB elements in their promoters. In mammals, IFN-beta transcription is induced through the RIG-I/MDA5 pathway where the adaptor protein IPS-1 plays a key role in the signal transduction. In this work we show that an Atlantic salmon homologue of IPS-1 (AsIPS-1) mediates activation of the salmon IFNa1 promoter and an NF-kappaB driven promoter. AsIPS-1 shares only 18% identity in amino acid sequence with human IPS-1, but possesses the CARD, proline-rich and transmembrane domains found in mammalian IPS-1. Overexpression of AsIPS-1 resulted in induction of an antiviral state in the cells apparently due to induction of IFN. Deletion of the CARD and transmembrane domains of AsIPS-1 abolished its ability to activate the IFNa1 promoter and the NF-kappaB driven promoter, and thus its ability to induce an antiviral state. AsIPS-1 is located to mitochondria similar to human IPS-1. Taken together, IPS-1 plays a key role in the induction of Atlantic salmon IFNa1, which appears to be the first and major IFN induced in host cells upon recognition of viral dsRNA.