Molecular cloning and characterisation of a fish PKR-like gene from cultured CAB cells induced by UV-inactivated virus

• Published in: Fish & shellfish immunology 2004-10, Vol.17 (4), p.353-366
• Main Authors: Hu, Cheng-Yu, Zhang, Yi-Bing, Huang, Guo-Ping, Zhang, Qi-Ya, Gui, Jian-Fang
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antiviral-relevant gene; Base Sequence; Blastula - cytology; Blastula - metabolism; CAB cell; Carassius auratus; Carps - genetics; Cells, Cultured; Cloning, Molecular; Cluster Analysis; Cycloheximide; DNA Primers; Double-stranded-RNA-dependent protein kinase; eIF-2 Kinase - biosynthesis; eIF-2 Kinase - genetics; Enzyme Induction - drug effects; Enzyme Induction - physiology; Expression; Grass carp haemorrhagic virus; Grass carp hemorrhage virus; Immune-relevant gene; Interferon; Interferons - metabolism; Interferons - pharmacology; Molecular Sequence Data; Phylogeny; Reoviridae; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Sequence Analysis, DNA; Ultraviolet Rays; Virus Inactivation
• ISSN: 1050-4648; 1095-9947
• DOI: 10.1016/j.fsi.2004.04.009

The double-stranded-RNA-dependent protein kinase (PKR) is an important component in an antiviral defence pathway that is mediated by interferon (IFN) in vertebrates. Previously, some important IFN system genes had been identified from an IFN-producing CAB (crucian carp Carassius auratus blastulae embryonic) cells after treatment with UV-inactivated GCHV (grass carp haemorrhage virus). Here, a fish PKR-like gene, named CaPKR-like, is cloned and sequenced from the same virally infected CAB cells. It has 2192 base pairs in length with a largest open reading frame (ORF) encoding a protein of 513 amino acid residues. BLAST search reveals that the putative CaPKR-like protein is most homologous to human PKR and also has a high-level homology with all members of a family of eIF2α kinases. Structurally, CaPKR-like possesses a conserved C-terminal catalytic domain of eIF2α kinase family and the most similarity to mammalian PKRs. Within its N-terminus, there are no dsRNA-binding domains conserved in mammalian PKRs instead of two putative Z-DNA binding domains (Zα). Like mammalian PKRs, CaPKR-like had a very low level of constitutive expression in normal CAB cells but was up-regulated in response to active GCHV, UV-inactivated GCHV and CAB IFN, implying that the transcriptional activation of CaPKR-like by viral infection is mediated possibly by newly produced CAB IFN, which was further supported by using cycloheximide, a potent inhibitor of protein synthesis. The results together suggested that CaPKR-like was the first identified fish gene most similar to mammalian PKRs.