Substrate specificity of three viral thymidine kinases (TK): vaccinia virus TK, feline herpesvirus TK, and canine herpesvirus TK.

In search of novel suicide gene candidates we have cloned and characterized thymidine kinases from three viruses; vaccinia virus TK (VVTK), feline herpesvirus TK (FHV-TK), and canine herpesvirus TK (CHV-TK). Our studies showed that VVTK primarily is a thymidine kinase, with a substrate specificity mainly restricted to dThd and only minor affinity for dCyd. VVTK also is related closely to mammalian thymidine kinase 1 (TK1), with 66% identity and 75% general homology. Although CHV-TK and FHV-TK are sequence related to herpes simplex virus types 1 thymidine kinase (HSV1-TK), with 31% and 35% identity and a general similarity of 54%, the substrate specificity of these enzymes was restricted to dThd and thymidine analogs.

Mutation hot spots in the canine herpesvirus thymidine kinase gene.

The guanine and cytosine content (GC-content) of alpha-herpesvirus genes are highly variable despite similar genome structures. It is known that drug resistant HSV, which has the genome with a high GC-content (approximately 70%), commonly includes frameshift mutations in homopolymer stretches of guanine (G) and cytosine (C) within the thymidine kinase (TK) gene. However, whether such mutation hotspots exist in the TK gene of canine herpesvirus (CHV) which has a low GC-content was unknown. In this study, we investigated mutations in the TK gene of CHV. CHV was passaged in the presence of iodo-deoxyuridine (IDU), and IDU-resistant clones were isolated. In all IDU-resistant virus clones, mutations in the TK gene were observed. The majority of these mutations were frameshift mutations of an adenine (A) insertion or deletion within either of 2 stretches of eight A's in the TK gene. It was demonstrated that CHV TK mutations frequently occur at a limited number of hot spots within long homopolymer nucleotide stretches.

Sequence of the canine herpesvirus thymidine kinase gene: taxon-preferred amino acid residues in the alphaherpesviral thymidine kinases.

Multiple sequence alignments of evolutionarily related proteins are finding increasing use as indicators of critical amino acid residues necessary for structural stability or involved in functional domains responsible for catalytic activities. In the past, a number of alignments have provided such information for the herpesviral thymidine kinases, for which three-dimensional structures are not yet available. We have sequenced the thymidine kinase gene of a canine herpesvirus, and with a multiple alignment have identified amino acids preferentially conserved in either of two taxons, the genera Varicellovirus and Simplexvirus, of the subfamily Alphaherpesvirinae. Since some regions of the thymidine kinases show otherwise elevated levels of substitutional tolerance, these conserved amino acids are candidates for critical residues which have become fixed through selection during the evolutionary divergence of these enzymes. Several pairs with distinctive patterns of distribution among the various viruses occur in or near highly conserved sequence motifs previously proposed to form the catalytic site, and we speculate that they may represent interacting, co-ordinately variable residues.

Identification and nucleotide sequence of the thymidine kinase gene of canine herpesvirus.

This paper presents the entire nucleotide sequence of the thymidine Kinase (TK) gene of canine herpesvirus (CHV). The gene was located within a 2.1 kbp EcoRV fragment by Southern-blot hybridization with a probe derived from the known feline herpesvirus type 1 (FHV-1) TK gene. An open reading frame (ORF) of 987 nucleotides, capable of encoding a TK translation product of 328 amino acids, was identified. Alignment of the predicted amino acid sequence of the CHV TK with other herpesvirus TKs revealed homologies of 25-47%. The proposed nucleotide-binding site and thymidine-binding site sequences of known herpesvirus TKs could be aligned with similar sequences in CHV TK. Northern-blot analysis revealed 1.3 kb and 5.0 kb mRNAs as the TK-specific transcripts. It is probable that the 1.3 kb transcript codes for the CHV TK and that the 5.0 kb transcript codes for the CHV TK and the downstream sequence.