Enzymatic basis for the selective inhibition of varicella-zoster virus by 5-halogenated analogues of deoxycytidine

Abstract

5-Bromodeoxycytidine (BrdC) and 5-iododeoxycytidine, at a concentration of 100 mug/ml, effectively inhibit the replication of varicella-zoster (VZ) virus in tissue culture. No toxicity could be demonstrated in uninfected cells under the same conditions. Studies on the enzymatic basis for this selective inhibition were undertaken. Infection of human embryonic lung cell monolayers with VZ virus-infected cells results in the induction of thymidine (dT), deoxycytidine (dC), and BrdC kinase activities (which are increased 10-, 40-, and 60-fold, respectively) and in a 70-fold stimulation in the incorporation of 3H nucleotide (5-bromodeoxyuridylate) derived from BrdC into DNA. The thermal stability of the VZ virus-induced activities differs significantly from the activities induced by herpes simplex virus type 1 and herpes simplex virus type 2 and those present in uninfected human embryonic lung cells. The VZ virus-induced dT, dC, and BrdC kinase are similarly affected by temperature and cofractionate upon Sephadex gel filtration, findings consistent with the hypothesis that these activities are the function of a single enzyme: a pyrimidine deoxyribonucleoside kinase. The molecular weight, calculated on the basis of the elution pattern on Sephadex G-150, is 70,000. Kinetic studies, demonstrating that dT and dC competively inhibit the phosphorylation of BrdC, are consistent with the phosphorylation of these substrates at a common active site. Kinetic parameters include: KidT = 0.6 MUM; KidC = 60 muM; KmBrdC = 8.5 muM. In contrast to its relatively high affinity for the VZ virus-induced kinase, BrdC is a relatively poor substrate for the host kinases. Therefore, the basis for the selective inhibition of VZ virus by 5-halogenated analogues of dC is reflected in the induction of a pyrimidine deoxyribonucleoside kinase with a high affinity for BrdC.

Induction of thymidine kinase and DNase in varicella-zoster virus-infected cells and kinetic properties of the virus-induced thymidine kinase

“…All enzyme activities examined were found to be induced in infected cells. This confirmed previous observations (9,11,18,19) that TK and DNA polymerase activities are induced in cells after VZV infection. In addition, DNase activities were about 20-fold greater in infected cells as compared with actively growing cells and about 7-fold greater than activities present in mock-infected cells.…”

“…Varicella-zoster virus (VZV) is one type of herpesvirus capable of causing human disease. Two enzyme activities, thymidine kinase (TK) (9,11,19) and DNA polymerase (18), have been shown to be induced in cultured cells after infection with this virus. Both induced enzymes seem to have properties different from the corresponding enzymes derived from host cells.…”

“…Both induced enzymes seem to have properties different from the corresponding enzymes derived from host cells. TK induced in infected human foreskin (HF) cells differed from the host TK not only in its physical properties but also in its substrate specificity when crude homogenates from infected and uninfected cells were compared (9,11,19,20). This raised the possibility of developing selective anti-VZV agents based on the unique viral TK, utilizing the same strategy as proposed previously for the development of specific anti-herpes simplex virus type 1 (HSV-1) and anti-HSV-2 agents (3).…”

Blocked herpes simplex virus type 2-specific DNA synthesis in simian virus 40-transformed hamster cells permissive for herpes simplex virus type 1

“…(ii) Kinase assay. The kinase assay used was a modification of that described by Doberson et al (6). Reaction mixtures consisted of the cell extract (0.05 ml) added to 0.05 ml of 50 mM Tris-hydrochloride buffer (pH 7.2) containing 5 mM ATP, 5 mM MgCl2, 10 mM NaF, and [3H]thymidine (21 M, 23 Ci/mmol).…”

Characterization of the Epstein-Barr virus-encoded thymidine kinase expressed in heterologous eucaryotic and procaryotic systems

“…All the human herpesviruses, except human cytomegalovirus, have been shown to induce a novel thymidine kinase (TK) activity in virus-infected cells (3,8).…”