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.
Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803.
L A
LA
Hanson
K G
KG
Kousoulas
R L
RL
Thune
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