Varicella-Zoster Virus Specifies a Thymidylate Synthetase

Abstract

SUMMARYA homology search of proteins predicted from the recently reported complete DNA sequence of varicella-zoster virus (VZV) revealed that the product of gene 13 was highly homologous to eukaryotic and prokaryotic thymidylate synthetases (TSs). The VZV protein was shown to be a TS by three functional tests. Firstly, a plasmid designed to express the native protein was able to complement a strain of Escherichia coil in which the natural TS gene is deleted. Secondly, in an enzyme assay for TS, extracts of the complemented strain were capable of releasing tritiated water from 2'-deoxy [5-3H]uridylate. Thirdly, these extracts contained a protein that bound isotopically labelled 5-fluoro-2'-deoxyuridylate, a ligand specific for the active site of TS. In addition, a novel ligand-binding protein was detected in human cells infected with VZV.Two common human diseases, chickenpox and shingles, are caused by varicella-zoster virus (VZV). This virus is a member of the Alphaherpesvirinae, a subfamily of the Herpesviridae typified by the most extensively studied human herpesvirus, herpes simplex virus type 1 (HSV-1). Davison & Scott (1986) identified genes encoding 67 unique proteins from an analysis of the complete sequence of the 125 kbp linear double-stranded DNA genome of VZV. Five glycoprotein genes were proposed from considerations of primary amino acid sequence, and the glycoprotein products of four have been detected (Ellis et al., 1985;Davison et al., 1985;Keller et al., 1986Keller et al., , 1987. Functional assignments for 12 additional VZV proteins are dependent on comparisons with available data for HSV-1 proteins of known function (Davison & Scott, 1986). The functions of two of the remaining 50 VZV proteins may be proposed on the basis of homology with non-herpesvirus proteins. One is related to several eukaryotic protein kinases (McGeoch & Davison, 1986), and the other is homologous to prokaryotic and eukaryotic thymidylate synthetases. In this paper, we describe the structural and functional identification of the VZV thymidylate synthetase (TS) gene and the detection of a novel TS in VZV-infected cells.TS (5,10-methylenetetrahydrofolate : dUMP C-methyltransferase, EC 2.1.1.45) is a homodimeric enzyme which catalyses the reductive methylation of deoxyuridylate to thymidylate. As the sole means for supplying thymidine nucleotides de novo, TS is crucial in DNA metabolism. A particularly striking feature of this enzyme is its high degree of amino acid sequence conservation. For example, the human and Escherichia coli proteins are 53% homologous (Takeishi et al., 1985). A computer-aided homology search of the 67 VZV proteins revealed that the product of gene 13 has a similar degree of homology to published TSs. The protein-coding t Present address:

Isolation of peptide aptamers that inhibit intracellular processes

“…Because a human thymidylate synthase gene can complement an E. coli thyA mutant (27), ABBIS could be used to identify peptide inhibitors of human thymidylate synthase as well. Similarly, thymidylate synthase genes from such diverse sources as Trypanosoma brucei (28), Cryptococcus neoformans (29), and varicella zoster virus (30) can complement an E. coli thyA mutant, raising the possibility of using ABBIS to develop inhibitors of thymidylate synthase from these important pathogens.…”

Varicella-zoster virus

“…The product of gene 13 bears strong homology to TS proteins from other organisms and several approaches have demonstrated that it is a functional TS (Thompson et al, 1987). Thus, VZV A. J. Davison supplements both pathways for providing thymidylate and it is possible that the dPyK and TS are required at different stages of virus pathogenesis.…”

“…The first herpesvirus genome to be sequenced was that of EBV (Baer et al, 1984) and a comparison of its gene products with those of VZV defined EBV homologues of 29 VZV genes (Davison & Taylor, 1987). The use of other criteria, such as gene location and orientation, suggested that several additional VZV genes might have EBV counterparts.…”

“…A inverted), B and C in EBV UL. Reprinted from Davison & Taylor (1987) with permission. contain many genes unique to each virus.…”

A Novel dCMP Methylase by Engineering Thymidylate Synthase

“…Finally, structural determinations of the TS N229D/H199A/dCMP and dUMP complexes confirmed that replacement of His 199 alleviates steric displacement of the pyrimidine from the position most suitable for catalysis. (10) and H199X (where X ) G, A, V, N, P, R) (13,15,16) were employed to construct six double mutants. DNA from each of the pSCTSH199X plasmids encoding the H199X mutants was digested with ApaI and BglII.…”