Efficacies of antiherpesvirus nucleosides against two strains of herpes simplex virus type 1 in Vero and human embryo lung fibroblast cells

Antimicrob Agents Chemother

Ishioka

Clercq

et al. 2003

As the number of patients treated with acyclovir (ACV) has increased, increasing numbers of ACV-resistant (ACV r ) herpes simplex virus (HSV) and varicella-zoster virus strains have been isolated, mainly from immunocompromised patients (8). The selectivity of ACV as an antiherpesvirus drug is based on its specific interaction with virus-encoded enzymes, thymidine kinase (TK) and DNA polymerase (DNA Pol). Therefore, the mechanisms responsible for ACV resistance are mutations in the TK and/or DNA Pol polypeptides (1). A previous largescale clinical study on ACV r HSV strains isolated from patients infected with human immunodeficiency virus indicated that 96% of ACV r HSV mutants were low producers of, or deficient in, TK activity (TK Ϫ ), with 4% being TK mutants with an altered substrate specificity. No DNA Pol mutants were isolated (12).It is generally believed that ACV r strains arise from naturally occurring mutations during DNA replication and are selected by ACV both in vitro (cell culture experiments) and in vivo (patients) (2). This hypothesis was formed from the following observations: (i) without exposure to ACV, approximately 0.3 to 20 ACV r mutants occur in 10 4 PFU of clinical HSV-1 isolates (11,19,26,28) and of a laboratory strain grown from one plaque (2); (ii) ACV r isolates under ACV selective pressure represent only a small proportion of the viable virions in the original virus population (19); and (iii) no ACV mutagenic activity has been detected in previous studies (3, 30).However, there is still a possibility that ACV does influence the development of ACV r strains during ACV treatment. To address the question of whether or not ACV induces mutation, we chose penciclovir (PCV), which is similar to ACV both in structure and in its need for phosphorylation by virus TK for its anti-HSV action, as a control drug and isolated a series of ACV r and PCV-resistant (PCV r ) strains emerging during serial passages of HSV-1 in the presence of ACV or PCV. Sequencing of the TK and DNA Pol genes showed differential mutation patterns in the ACV r and PCV r isolates, suggesting that one, if not both, of the drugs had a differential effect on the generation of drug-resistant mutants. MATERIALS AND METHODS Cells and viruses.A human osteosarcoma TK-deficient cell line, 143B/ TK Ϫ neo R , was kindly supplied by Riken Cell Bank, Tsukuba, Japan. Human embryo lung (HEL) fibroblasts, Vero, and 143B/TK Ϫ neo R cells were cultivated in Eagle's minimum essential medium (MEM) supplemented with 10% calf serum. The VR-3 strain of HSV-1 described previously (6) was obtained from the American Type Culture Collection, Rockville, Md. This virus strain was plaque purified three times, grown in HEL cells for less than three passages, and stored in small portions at Ϫ80°C.Isolation of drug-resistant viruses. ACV r and PCV r HSV-1 strains were isolated by serial passage of the reference VR-3 strain in the presence of increasing concentrations of ACV or PCV, as follows. Twenty PFU of the VR-3 strain were inoculated onto HEL cell mon...

“…The susceptibilities of virus clones to antiherpesvirus compounds were assayed with a 50% plaque reduction assay in Vero cells, as described previously (31).…”

Section: Cells and Virusesmentioning

confidence: 99%

Differential Mutation Patterns in Thymidine Kinase and DNA Polymerase Genes of Herpes Simplex Virus Type 1 Clones Passaged in the Presence of Acyclovir or Penciclovir

Antimicrob Agents Chemother

Ishioka

Clercq

et al. 2003

“…The VR-3 strain of HSV-1 and the UW-268 strain of HSV-2 were generously supplied by the American Type Culture Collection, Rockville, Md. The methods for the preparation of the stock viruses have been described previously (13). The ACV-resistant mutants of strains VR-3 and UW-268 (referred to as strains VRTK-and UWTK-, respectively) were isolated as follows.…”

Section: Methodsmentioning

confidence: 99%

“…The effects of the antiviral compounds on the replication of HSV were evaulated by a plaque reduction method as described previously (13).…”

Section: Methodsmentioning

confidence: 99%

Effects of various nucleosides on antiviral activity and metabolism of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil against herpes simplex virus types 1 and 2

Antimicrob Agents Chemother

Machida

Sakuma

et al. 1988

Self Cite

Uptake of 1-(3-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) into herpes simplex virus type 1 (HSV-1)-and 2 (HSV-2)-infected cells was elevated about 190 to 40 times, compared with that into mock-infected human embryo lung fibroblast cells. Uptake was not enhanced by infection with thymidine kinase-negative HSV-1 and HSV-2 mutants, however. In HSV-1-infected cells, 9.7% of BV-araU was phosphorylated to BV-araU triphosphate, but only 1.1% was phosphorylated in HSV-2-infected cells. The antiviral effect, uptake, and turnover of BV-araU were inhibited significantly by thymidine (dThd), moderately by deoxyuridine, and not at all by deoxycytidine. On the other hand, the antiviral activity of acyclovir (ACV) was inhibited only by dThd. The effect of BV-araU was influenced by dThd and dThd phosphates (mono-, di-, and triphosphates), and the effect of ACV was influenced only by dThd, which competitively inhibited the phosphorylation of ACV to ACV monophosphate. The combination of 5-fluorodeoxyuridine (FUdR)-BV-araU or FUdR-ACV had a synergistic effect on HSV-1 and HSV-2 replication. The effect of FUdR on the turnover of BV-araU in HSV-1-and HSV-2-infected cells was analyzed by high-performance liquid chromatography. In HSV-1-infected cells, 86% of the BV-araU was phosphorylated to BV-araU triphosphate, but no effect was observed in HSV-2-infected cells, in which 98% of the BV-araU remained as BV-araU monophosphate.

“…Tables and Fig. 2. The products of the enzyme reaction were separated by adsorption onto DE81 discs (Whatman) or by PEI-cellulose thin-layer chromatography (Macherey-Nagel) and radioactivity was measured in a Beckman scintillation counter as described previously (Suzutani et al, 1988a).…”

Section: Materials and Chemicalsmentioning

confidence: 99%

Kinetic studies of the predicted substrate-binding site of varicella-zoster virus thymidine kinase

Journal of General Virology

Davies

Honess

1993

To investigate the mechanism of kinetic action and substrate recognition of varicella-zoster virus (VZV) thymidine kinase (TK), we designed and isolated a sitedirected mutant VZV TK which has double amino acid substitutions, 136threonine to leucine and la7isoleucine to leucine (SDM TK). This mutant was designed to alter the substrate-binding site of the VZV TK to duplicate that of the herpes simplex virus type 2 enzyme. Kinetic studies of the activity of wild-type TK indicated that the binding order of ATP and thymidine is random and that wild-type VZV TK possessed high thymidylate kinase (TM-K) activity. The sensitivity of VZV TK to bisubstrate analogues, dinucleotides of adenosine and thymidine, showed that the optimum distance between the ATP-and substrate-binding sites is two phosphoryl groups greater than with the natural substrate for TK activity. SDM TK lost deoxycytidine kinase activity and had reduced TK and TM-K activities. Inhibition studies on both WT and SDM TK by 5-halogenovinyluridine analogues and their 5' monophosphate derivatives revealed that amino acids at positions 136 and 137 are involved in substrate binding, probably through a role in the formation of the binding pocket for bulky substrates.