Molecular cloning and characterisation of a fish PKR-like gene from cultured CAB cells induced by UV-inactivated virus.
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 eIF2alpha kinases. Structurally, CaPKR-like possesses a conserved C-terminal catalytic domain of eIF2alpha 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 (Zalpha). 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.
State Key Laboratory of Freshwater Ecology and Biotechnology, Wuhan Center for Developmental Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Cheng-Yu
CY
Hu
Yi-Bing
YB
Zhang
Guo-Ping
GP
Huang
Qi-Ya
QY
Zhang
Jian-Fang
JF
Gui
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D036703:Blastula / Q000166:cytology / Q000378:metabolism; D002347:Carps / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016000:Cluster Analysis; D003513:Cycloheximide; D017931:DNA Primers; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D007372:Interferons / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D010802:Phylogeny; D012087:Reoviridae; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014466:Ultraviolet Rays; D038301:Virus Inactivation; D019892:eIF-2 Kinase / Q000096:biosynthesis* / Q000235:genetics*
D017931:DNA Primers; D007372:Interferons; D003513:Cycloheximide; D019892:eIF-2 Kinase