Characterization of a varicella-zoster virus variant with altered thymidine kinase activity.

ABSTRACT Varicella-zoster virus (VZV) is a common herpesvirus responsible for disseminated or chronic infections in immunocompromised patients. Effective drugs such as acyclovir (ACV), famciclovir (prodrug of penciclovir), and foscarnet are available to treat these infections. Here we report the phenotypic and genetic characterization of four ACV-resistant VZV strains isolated from AIDS patients and transplant recipients. Sensitivity to six antiviral drugs was determined by an enzyme-linked immunosorbent assay, viral thymidine kinase (TK) activity was measured by comparing [3H]thymidine and 1-β-d-arabinofuranosyl-[3H]thymine as substrates, and the TK gene open reading frame was sequenced. Three strains were found to be TK deficient, and the fourth was a mixed population composed of TK-positive and TK-deficient viruses. Each strain presented a unique TK gene mutation that could account for ACV resistance. In one strain, the deletion of two nucleotides at codon 215 induced a premature stop signal at codon 217. In another strain, a single nucleotide addition at codon 167 resulted in a premature stop signal at codon 206. In both other strains, we identified amino acid substitutions already described in other ACV-resistant VZV strains: either Glu→Gly at residue 48 or Arg→Gly at residue 143. According to our work and data previously reported on resistant VZV strains, there are three areas in the TK gene where 71% of the mutations described to date are located. These areas are putative candidates for a genotypic diagnosis of ACV resistance.

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Varicella-Zoster Virus ORF47 Protein Serine Kinase: Characterization of a Cloned, Biologically Active Phosphotransferase and Two Viral Substrates, ORF62 and ORF63

Journal of Virology ◽

10.1128/jvi.75.18.8854-8858.2001 ◽

2001 ◽

Vol 75 (18) ◽

pp. 8854-8858 ◽

Cited By ~ 58

Author(s):

T. K. Kenyon ◽

J. Lynch ◽

J. Hay ◽

W. Ruyechan ◽

C. Grose

Keyword(s):

Varicella Zoster Virus ◽

Kinase Activity ◽

Biologically Active ◽

Regulatory Proteins ◽

Kinase Assay ◽

Serine Kinase ◽

Varicella Zoster ◽

Simplex Virus

ABSTRACT Varicella-zoster virus (VZV) codes for a protein serine kinase called ORF47; the herpes simplex virus (HSV) homolog is UL13. No recombinant alphaherpesvirus serine kinase has been biologically active in vitro. We discovered that preservation of the intrinsic kinase activity of recombinant VZV ORF47 required unusually stringent in vitro conditions, including physiological concentrations of polyamines. In this assay, ORF47 phosphorylated two VZV regulatory proteins: the ORF62 protein (homolog of HSV ICP4) and the ORF63 protein (homolog of HSV ICP22). Of interest, ORF47 kinase also coprecipitated ORF63 protein from the kinase assay supernatant.

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Characterization of virulent and attenuated strains of pseudorabies virus for thymidine kinase activity, virulence and restriction patterns

10.31274/rtd-180816-770 ◽

1986 ◽

Author(s):

Michael Dean McFarland

Keyword(s):

Thymidine Kinase ◽

Pseudorabies Virus ◽

Kinase Activity ◽

Thymidine Kinase Activity ◽

Restriction Patterns

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β-L-1-[5-(E-2-Bromovinyl)-2-(hydroxymethyl)-1,3-dioxolan-4-yl)] uracil (L-BHDU) effectiveness against varicella-zoster virus and herpes simplex virus type 1 depends on thymidine kinase activity

10.1101/2020.02.13.948190 ◽

2020 ◽

Author(s):

Chandrav De ◽

Dongmei Liu ◽

Daniel Depledge ◽

Judith Breuer ◽

Uma S. Singh ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Thymidine Kinase ◽

Herpes Simplex Virus Type ◽

Kinase Activity ◽

Wild Type ◽

Thymidine Kinase Activity ◽

Varicella Zoster ◽

Simplex Virus

Abstractß-L-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (L-BHDU) prevents varicella-zoster virus (VZV) replication in cultured cells and in vivo. Its mechanism of action was investigated by evaluating its activity against related herpesviruses and by analyzing resistant VZV strains. L-BHDU was effective against herpes simplex virus type 1 (HSV-1) with an EC50 of 0.22 µM in human foreskin fibroblast (HFF) cells. L-BHDU also inhibited HSV-2 and simian varicella virus (SVV) to a lesser extent. VZV mutants resistant to L-BHDU and other antiviral compounds were obtained by serial passage of the wild type VZV pOka and VZV Ellen strains in the presence of increasing drug concentrations. VZV strains resistant to L-BHDU (L-BHDUR) were cross-resistant to acyclovir (ACV) and brivudin (BVdU) but not to foscarnet (PFA) and cidofovir (CDV). Conversely, ACV-resistant strains were also resistant to L-BHDU. Whole genome sequencing of L-BHDUR strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV thymidine kinase (TK). The wild type and mutant forms of VZV TK were cloned as GST fusion proteins and expressed in E. coli. The partially purified TKG22R-GST and TKR130Q- GST proteins failed to convert thymidine to thymidine monophosphate whereas the wild type TK-GST protein was enzymatically active. Similarly, L-BHDUR virus TK did not phosphorylate the drug. As expected, wild type VZV converted L-BHDU to L-BHDU monophosphate and diphosphate forms. In conclusion, L-BHDU effectiveness against VZV and HSV-1 depends on thymidine kinase activity.

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