Channel catfish herpesvirus (CCV) encodes a functional thymidine kinase gene: elucidation of a point mutation that confers resistance to Ara-T.
The channel catfish herpesvirus (CCV) thymidine kinase (TK) gene was mapped on the CCV genome by marker rescue analysis using a TK-deficient channel catfish ovary cell line (CCO), a TK-negative CCV mutant, and a panel of cloned CCV genomic DNA fragments. The TK-deficient cell line (CCOBr) was isolated after repeated propagation of CCO cells in increasing concentrations of 5-bromo-2'-deoxyuridine. Infection of CCOBr cells with CCV produced high levels of TK activity. The TK- virus (CCVAr) was isolated after repeated propagation in the presence of the TK-activated antiherpetic agent, 1-beta-D-arabinofuranosylthymine (Ara-T). A CCV genomic DNA library was constructed into cosmid pHC 79. Marker rescue analysis mapped the mutation within a 3.1-kb fragment located internal to the 18-kb repeat ends of the CCV genome. These genomic coordinates contained a putative TK gene identified by homology to other herpesvirus TK and cellular deoxycytidine kinase genes. DNA sequencing of the mapped coordinates identified the presence of a single mutation in the CCVAr mutant virus which resulted in a stop codon at amino acid position 97. These results functionally confirm that ORF 5 identified by Davison (Virology 186, 9-14, 1992) is the TK gene and show that CCV is amenable to marker rescue and marker transfer genetic analyses extensively used for investigations of the molecular biology of other herpesviruses.
Hanson
L A
LA
Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803.
Kousoulas
K G
KG
Thune
R L
RL
D000818:Animals; D001087:Arabinonucleosides / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial; D005814:Genes, Viral; D006564:Herpesviridae / Q000201:enzymology* / Q000235:genetics; D007059:Ictaluridae / Q000382:microbiology*; D008969:Molecular Sequence Data; D009154:Mutation; D012091:Repetitive Sequences, Nucleic Acid; D013936:Thymidine / Q000031:analogs & derivatives / Q000494:pharmacology; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection; D015678:Viral Structural Proteins / Q000235:genetics
D001087:Arabinonucleosides; D015678:Viral Structural Proteins; D013937:Thymidine Kinase; C010032:thymine arabinoside; D013936:Thymidine