Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sergerie, Y. Articles by Boivin, G. Search for Related Content PubMed PubMed Citation Articles by Sergerie, Y. Articles by Boivin, G.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, November 2006, p. 3889-3892, Vol. 50, No. 11
0066-4804/06/$08.00+0     doi:10.1128/AAC.00889-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Yan Sergerie and Guy Boivin^*

Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, QC, Canada

Received 18 July 2006/ Returned for modification 8 August 2006/ Accepted 5 September 2006

Top Abstract Text References

 
Contributions of thymidine kinase (TK) mutations to acyclovir (ACV) resistance were evaluated in herpes simplex virus type 1 recombinant viruses generated using a set of overlapping cosmids and plasmids. Alterations in both conserved and nonconserved regions of the TK gene were shown to confer high levels of resistance to ACV.

Top Abstract Text References

 
Herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) infections are associated with serious complications in immunocompromised hosts. Prolonged antiviral treatment is often required for the clinical management of these patients, which could lead to the emergence of drug-resistant viruses (5, 17). Acyclovir (ACV), which has been the standard treatment for HSV infections, must be phosphorylated first by the viral thymidine kinase (TK) and then by cellular kinases before inhibiting the virus-encoded DNA polymerase (DNA pol) (reviewed in reference 7). In the clinic, HSV resistance to ACV is most commonly due to mutations (substitutions, deletions, or additions) in the viral TK gene (6). Single base substitutions in the viral DNA pol gene have been more rarely reported in ACV-resistant clinical HSV isolates, with or without additional TK mutations (14, 17). Because of the presence of multiple mutations within these two genes including several ones associated with viral polymorphism (2, 9-11, 13, 17), it has been difficult so far to analyze the contribution of each specific HSV mutation with regard to the ACV resistance phenotype. We recently reported the generation and characterization of HSV-1 DNA pol mutants (1) using a set of four overlapping cosmids and three plasmids based on the original work of Cunningham and Davison (3). Herein, we report a modification of this cosmid-based recombination system to evaluate the impact of HSV-1 TK mutations as well as double TK-DNA pol mutations on the ACV resistance phenotype.

Vero cells were maintained in minimum essential medium supplemented with 10% fetal calf serum. The HSV-1 recombinant strain 17 and all mutant viruses derived from this strain were propagated in Vero cells. The method used to generate HSV-1 recombinant viruses from overlapping cosmids (gift of Charles Cunningham, MRC Virology Unit, Glasgow, United Kingdom) as well as the plasmid used for site-directed mutagenesis of the viral DNA pol gene have been described elsewhere (1). Additional modifications were made to this system to introduce TK mutations as follows. One of the five viral fragments (cosmid 71, corresponding to nucleotides [nt] 40966 to 77049) was first replaced by three overlapping plasmids (pNEB23, pPol6, and pNEB10) (1) (Fig. 1). Plasmid pYS-1 was subsequently created by inserting the purified pNEB23 BbvcI-MfeI-digested fragment (nt 43981 to 49891) into plasmid pSP72 (Promega, Madison, WI), in which BbvcI-MfeI restriction sites had been added (Fig. 1). Specific mutations in the TK gene (located between nt 186 and nt 1007 of the viral gene) were introduced into plasmid pYS-1 using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mutated BbvcI-MfeI fragments were then cloned back into pNEB23. The correct nucleotide sequence of each mutated TK gene was verified by sequence analysis and compared to that of strain 17. Mutations were introduced in the viral DNA pol gene by performing site-specific mutagenesis on plasmid pPol6 as reported previously (1).

View larger version (24K): [in this window] [in a new window]

FIG. 1. Schematic representation of viral cosmids and plasmids used to generate HSV-1 recombinants into Vero cells. (A) Digested fragments of five cosmids (24, 32, 48, 51, and 71) covering the complete genome of HSV-1 strain 17 (3) with three plasmids (pNEB23, pPol6, and pNEB10) designed to replace cos 71. (B and C) Construction of plasmids pNEB23, pPol6, and pNEB10 (B) and pYS-1 (C).

Generation of HSV-1 TK and/or DNA pol mutants, antiviral susceptibility assays, and in vitro replication kinetic experiments were done as previously described (1). Briefly, digested cosmids (n = 4) and plasmids (n = 3) were transfected using Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada) into Vero cells seeded in 12-well plates. Viral cytopathic effects were observed approximately 3 to 4 days after transfection. The TK and DNA pol sequences of each recombinant virus were again verified by DNA sequencing. The acyclovir susceptibility of each recombinant virus was determined by the use of a plaque reduction assay (15). A twofold difference in 50% inhibitory concentration (IC₅₀) values between the recombinant wild-type (WT) and mutant viruses was considered significant (1). All experiments were done in duplicate using two independently generated viruses for each mutation of interest. Replication kinetics of each recombinant mutant virus (tested in duplicate at a multiplicity of infection of 2) were compared to those of the recombinant WT virus by titrating extracellular viruses collected at specific times postinfection (up to 120 h) onto Vero cells (1).

Eight recombinant HSV-1 viruses (WT, TK-deleted, four TK mutants, one DNA pol mutant, and one dual TK-DNA pol mutant), selected on the basis of their report in previous clinical or laboratory studies, were successfully generated in this study. Acyclovir IC₅₀ values are reported in Table 1. All recombinant mutants were resistant to ACV with the single DNA pol mutant D907V being the least resistant (fourfold change in IC₅₀ compared to WT) and the double TK (deleted C467 with truncated protein)-DNA pol (D907V) mutant being the most resistant (62-fold change in IC₅₀). As shown in Fig. 2, the replication kinetics of the mutants were relatively similar to those of the WT virus up to 120 h postinfection.

View this table: [in this window] [in a new window]

TABLE 1. Phenotypic and genotypic analyses of thymidine kinase and/or DNA polymerase HSV-1 recombinant mutantsa

View larger version (26K): [in this window] [in a new window]

FIG. 2. Replication kinetics of several HSV-1 mutants and the WT virus in a single-step growth experiment. On the day of infection, confluent Vero cells in 12-well plates were infected with recombinant viruses at a multiplicity of infection of 2. Extracellular viruses were collected at different times postinfection (2, 18, 24, 36, 48, 72, 96, and 120 h) and titrated onto Vero cells. Curves represent means of two independent experiments. (A) WT and single TK recombinant mutants. (B) WT and dual TK-DNA pol recombinant mutants (alone and in combination).

Although the definition of ACV resistance used in this study was somewhat arbitrary (2-fold change in IC₅₀ compared to WT), our results are in agreement with the ACV resistance phenotype previously reported in other studies that most commonly used a resistance cutoff of 2 µg/ml for clinical isolates (6, 17). Our results obtained with recombinant viruses, which allow mutational studies in a homogeneous background, revealed that several mutations in conserved but also in nonconserved regions of the TK gene are associated with high levels of ACV resistance. The mutation K62N, located in the ATP-binding site of the TK, conferred the highest level of resistance to ACV with the exception of the TK-deleted virus and the dual TK-DNA pol mutant. The importance of the lysine at position 62 has been previously demonstrated in a site-directed mutagenesis study (12). This mutation, also described in an ACV-resistant varicella-zoster virus isolate (19), has been associated with a TK-low-producer phenotype. The mutation C336Y, implicated in the conformation of the TK protein, also resulted in a phenotype highly resistant to ACV. The latter mutation was first described in laboratory-derived ACV-resistant mutants (4, 8) but also in a TK-altered clinical HSV-2 isolate (16). Of interest, the mutation P131S, located in a nonconserved region of the TK gene, also conferred an ACV-resistant 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. A single TK mutation (deletion of a C at position 467 leading to a truncated protein) induced a high level of ACV resistance whereas the single DNA pol mutation D907V, within a nonconserved gene region, induced a low level of resistance to ACV. The recombinant virus containing the two previous mutations had the highest ACV IC₅₀ value among all recombinant viruses, confirming the synergistic effect of dual TK-DNA pol mutations. The simultaneous presence of these two mutations was reported in an HSV-2 isolate from a patient with AIDS in whom ACV and foscarnet therapy sequentially failed (17). Our study unequivocally shows that the deletion of a C at position 467 in a homopolymer run, which introduces a stop codon 25 amino acids downstream, was mainly responsible for the ACV resistance phenotype of this isolate. Finally, as expected, the TK-deleted recombinant virus was highly resistant to ACV although not much more than single TK substitutions such as K62N and C336Y.

Alterations in the TK are most frequently seen in the clinic, probably because this protein is not essential for viral replication in most tissues and cultured cells (6, 13). Thus, it is not expected that mutations in this gene would affect viral growth kinetics. Indeed, the replication kinetics of all tested recombinant viruses including the TK-deleted virus were similar to those of the WT virus. In addition, mutation D907V, located in a nonconserved region of the DNA pol gene, did not affect viral replication, in contrast to other previously reported DNA pol mutations located within conserved gene regions (1).

In conclusion, we have developed a system that now allows rapid assessment of any mutations found in both viral genes associated with ACV resistance. Such methodology could serve for development of virtual drug phenotypes for HSV with systematic evaluation of viral mutations found in clinical specimens.

 
* Corresponding author. Mailing address: CHUQ-CHUL, room RC-709, 2705, blvd Laurier, Sainte-Foy, Québec, Canada G1V 4G2. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail: guy.boivin{at}crchul.ulaval.ca.

Published ahead of print on 18 September 2006.

Top Abstract Text References

 

1
1. Bestman-Smith, J., and G. Boivin. 2003. Drug resistance patterns of recombinant herpes simplex virus DNA polymerase mutants generated with a set of overlapping cosmids and plasmids. J. Virol. 77:7820-7829.[Abstract/Free Full Text]
2
2. Bestman-Smith, J., I. Schmit, B. Papadopoulou, and G. Boivin. 2001. Highly reliable heterologous system for evaluating resistance of clinical herpes simplex virus isolates to nucleoside analogues. J. Virol. 75:3105-3110.[Abstract/Free Full Text]
3
3. Cunningham, C., and A. J. Davison. 1993. A cosmid-based system for constructing mutants of herpes simplex virus type 1. Virology 197:116-124.[CrossRef][Medline]
4
4. Darby, G., B. A. Larder, and M. M. Inglis. 1986. Evidence that the "active centre" of the herpes simplex virus thymidine kinase involves an interaction between three distinct regions of the polypeptide. J. Gen. Virol. 67:753-758.[Abstract/Free Full Text]
5
5. Erlich, K. E., J. Mills, P. Chatis, G. J. Mertz, D. F. Busch, S. E. Follansbee, R. M. Grant, and C. S. Crumpacker. 1989. Acyclovir-resistant herpes simplex virus infections in patients with the acquired immunodeficiency syndrome. N. Engl. J. Med. 320:293-296.[Medline]
6
6. Gaudreau, A., E. Hill, H. H. Balfour, Jr., A. Erice, and G. Boivin. 1998. Phenotypic and genotypic characterization of acyclovir-resistant herpes simplex viruses from immunocompromised patients. J. Infect. Dis. 178:297-303.[Medline]
7
7. Gilbert, C., J. Bestman-Smith, and G. Boivin. 2002. Resistance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms. Drug Resist. Updates 5:88-114.[CrossRef][Medline]
8
8. Hwang, C. B., and H. J. Chen. 1995. An altered spectrum of herpes simplex virus mutations mediated by an antimutator DNA polymerase. Gene 152:191-193.[CrossRef][Medline]
9
9. Knopf, C. W., and K. Weisshart. 1988. The herpes simplex virus DNA polymerase: analysis of the functional domains. Biochim. Biophys. Acta 951:298-314.[Medline]
10
10. Kudo, E., H. Shiota, T. Naito, K. Satake, and M. Itakura. 1998. Polymorphisms of thymidine kinase gene in herpes simplex virus type 1: analysis of clinical isolates from herpetic keratitis patients and laboratory strains. J. Med. Virol. 56:151-158.[CrossRef][Medline]
11
11. Lee, N. Y., Y. Tang, M. J. Espy, C. P. Kolbert, P. N. Rys, P. S. Mitchell, S. P. Day, S. L. Henry, D. H. Persing, and T. F. Smith. 1999. Role of genotypic analysis of the thymidine kinase gene of herpes simplex virus for determination of neurovirulence and resistance to acyclovir. J. Clin. Microbiol. 37:3171-3174.[Abstract/Free Full Text]
12
12. Liu, Q., and W. C. Summers. 1988. Site-directed mutagenesis of a nucleotide-binding domain in HSV-1 thymidine kinase: effects on catalytic activity. Virology 163:638-642.[CrossRef][Medline]
13
13. Morfin, F., G. Souillet, K. Bilger, T. Ooka, M. Aymard, and D. Thouvenot. 2000. Genetic characterization of thymidine kinase from acyclovir-resistant and susceptible herpes simplex virus type 1 isolated from bone marrow transplant recipients. J. Infect. Dis. 182:290-293.[CrossRef][Medline]
14
14. Sacks, S. L., R. J. Wanklin, D. E. Reece, K. A. Hicks, K. L. Tyler, and D. M. Coen. 1989. Progressive esophagitis from acyclovir-resistant herpes simplex. Clinical roles for DNA polymerase mutants and viral heterogeneity? Ann. Intern. Med. 111:893-899.[Abstract/Free Full Text]
15
15. Safrin, S., S. Kemmerly, B. Plotkin, T. Smith, N. Weissbach, D. De Veranez, L. D. Phan, and D. Cohn. 1994. Foscarnet-resistant herpes simplex virus infection in patients with AIDS. J. Infect. Dis. 169:193-196.[Medline]
16
16. Sasadeusz, J. J., F. Tufaro, S. Safrin, K. Schubert, M. M. Hubinette, P. K. Cheung, and S. L. Sacks. 1997. Homopolymer mutational hot spots mediate herpes simplex virus resistance to acyclovir. J. Virol. 71:3872-3878.[Abstract]
17
17. Schmit, I., and G. Boivin. 1999. Characterization of the DNA polymerase and thymidine kinase genes of herpes simplex virus isolates from AIDS patients in whom acyclovir and foscarnet therapy sequentially failed. J. Infect. Dis. 180:487-490.[CrossRef][Medline]
18
18. Suzutani, T., K. Ishioka, E. De Clercq, K. Ishibashi, H. Kaneko, T. Kira, K. Hashimoto, M. Ogasawara, K. Ohtani, N. Wakamiya, and M. Saijo. 2003. 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. 47:1707-1713.[Abstract/Free Full Text]
19
19. Talarico, C. L., W. C. Phelps, and K. K. Biron. 1993. Analysis of the thymidine kinase genes from acyclovir-resistant mutants of varicella-zoster virus isolated from patients with AIDS. J. Virol. 67:1024-1033.[Abstract/Free Full Text]

---

Antimicrobial Agents and Chemotherapy, November 2006, p. 3889-3892, Vol. 50, No. 11
0066-4804/06/$08.00+0     doi:10.1128/AAC.00889-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sergerie, Y. Articles by Boivin, G. Search for Related Content PubMed PubMed Citation Articles by Sergerie, Y. Articles by Boivin, G.