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

Abstract

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...

Thymidine Kinase Mutations Conferring Acyclovir Resistance in Herpes Simplex Type 1 Recombinant Viruses

“…Of interest, the mutation P131S, located in a nonconserved region of the TK gene, also conferred an ACVresistant phenotype. This mutation has been previously described in a laboratory-derived mutant passaged in the presence of ACV (18). Since this ACV-resistant mutant contained both the P131S TK mutation and a DNA pol mutation (A719V), our study was able to confirm the role of the former mutation in conferring resistance to ACV.…”

Genotypic Characterization of UL23 Thymidine Kinase and UL30 DNA Polymerase of Clinical Isolates of Herpes Simplex Virus: Natural Polymorphism and Mutations Associated with Resistance to Antivirals

“…To date, the latter amino acid change has been reported only in an HSV-1 laboratory strain selected after serial passages in the presence of ACV. But this strain harbored mutations in both TK and DNA polymerase, making it difficult to interpret the respective roles of each mutation regarding ACV resistance (52). Moreover, the changes R176Q for HSV-1 and R177W for HSV-2 have been associated with ACV resistance (21,24,30,39).…”

In Vitro -Selected Drug-Resistant Varicella-Zoster Virus Mutants in the Thymidine Kinase and DNA Polymerase Genes Yield Novel Phenotype-Genotype Associations and Highlight Differences between Antiherpesvirus Drugs

“…In conclusion, the more complete DNA chain termination activity of PCV-TP, the weaker interaction with DNA Pol than ACV-TP, and the lack of homopolymeric stretches of Gs in the VZV TK and Pol genes may explain the differences observed between ACV and PCV in the present study. It is worth mentioning that different mutational patterns in TK genes of HSV were reported with viruses selected in cell culture for resistance to ACV or PCV (50,60). Additionally, PCV, similar to GCV, may be phosphorylated, not only by VZV TK, but also by the protein kinase ORF47, which is homologous to the HCMV protein kinase UL97.…”