Identification and analysis of the simian varicella virus thymidine kinase gene.
The thymidine kinase (TK) of herpesviruses, in contrast to cellular TKs, phosphorylates a variety of substrates including antiherpetic nucleoside analogues. This study reports the identification and DNA sequence of the simian varicella virus (SVV) TK gene. A 32P-labeled varicella zoster virus (VZV) TK DNA probe hybridized to the HindIII B subclone of the SVV BamHI B restriction endonuclease (RE) fragment, indicating the presence of a SVV DNA sequence homologous to the VZV TK gene. DNA sequence analysis of the SVV HindIII B subclone revealed a 1014 base pair (bp) open reading frame (ORF) encoding a 337 amino acid polypeptide homologous to herpesvirus TKs. The predicted SVV and VZV TK polypeptides share 51.3% identity, and alignment of the putative protein sequence of several TK homologues suggests the position of a conserved nucleotide binding site and a nucleoside (substrate) binding site in the SVV TK. Identification of the 5' end of the SVV TK transcript by primer extension analysis allowed a comparison of the SVV and VZV TK promoter regions indicating extensive conservation of the DNA sequence and transcription factor binding sites. Plaque reduction assays demonstrate that the SVV TK is active based on the susceptibility of SVV to acyclovir treatment and that SVV is less sensitive to acyclovir than VZV and herpes simplex virus (HSV-1) in infected Vero cells. Identification of the SVV TK ORF will facilitate studies that examine the role of viral TKs in pathogenesis and antiviral sensitivity and provides a potential insertion site for the expression of foreign genes.
Pumphrey
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Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, USA.
Gray
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D000212:Acyclovir / Q000633:toxicity; D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000633:toxicity; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D017931:DNA Primers; D015245:Deoxyribonuclease BamHI; D005814:Genes, Viral; D016679:Genome, Viral; D006568:Herpesvirus 1, Cercopithecine / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics*; D018140:Varicellovirus / Q000201:enzymology / Q000235:genetics*; D010948:Viral Plaque Assay; D015678:Viral Structural Proteins / Q000235:genetics*
D000998:Antiviral Agents; D017931:DNA Primers; D015678:Viral Structural Proteins; D013937:Thymidine Kinase; D015245:Deoxyribonuclease BamHI; D000212:Acyclovir