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Contribution of thymidine kinase (TK) mutations to acyclovir (ACV) resistance were evaluated in herpes simplex type 1 recombinant viruses generated using a set of overlapping cosmids and plasmids. Figure 1: Schematic representation of viral cosmids and plasmids used to generate HSV-1 recombinants into Vero cells. Figure 2: Replication kinetics of several HSV-1 mutants and the WT virus in a single-step growth experiment. Examination of the tCUP cryptic promoter from tobacco demonstrates that cryptic gene regulatory elements in the plant genome are functionally equivalent to elements responsible for the expression of plant genes. JavaScript is currently disabled, this site works much better if you enable JavaScript in your browser.
Alterations in both conserved and non-conserved regions of the TK gene were shown to confer high levels of resistance to ACV.Herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) infections are associated with serious complications in immunocompromised hosts. On the day of infection, confluent Vero cells in 12-well plates were infected with recombinant viruses at a MOI of 2. Prolonged antiviral treatment is often required for the clinical management of these patients, which could lead to the emergence of drug-resistant viruses [222, 230]. Extracellular viruses were collected at different times post-infection (up to 120 h) and titrated onto Vero cells. Analysis of the expression pattern of the GUS reporter gene in transgenic Arabidopsis plants revealed that all of the information needed for strong constitutive expression was located in the truncated, -394tCUP promoter fragment. Acyclovir (ACV), which has been the standard treatment for HSV infections, must be first phosphorylated by the viral thymidine kinase (TK) and then by cellular kinases before inhibiting the virus-encoded DNA polymerase (DNA pol) (reviewed in [442]).
Drug resistance patterns of recombinant herpes simplex virus DNA polymerase mutants generated with a set of overlapping cosmids and plasmids.
A series of 5? deletion and linker-scan mutagenesis constructs identified two separate enhancer elements.
In clinic, HSV resistance to ACV is most commonly due to mutations (substitutions, deletions or additions) in the viral TK gene [233]. A long AT-rich region was identified between positions ?350 and ?161 bp relative to the transcription start site. 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 [230, 286]. Because of the presence of multiple mutations within these two genes including several ones associated with viral polymorphism [230, 234, 249, 261, 446, 447], it has been difficult so far to analyze the contribution of each specific HSV mutation with regard to the ACV resistance phenotype. A 21 bp sequence (TAGCCCCAATTTCAAATTCAA) spanning nucleotides ?150 to ?130 relative to transcription start site was also identified in a similar fashion and defined a novel cryptic constitutive enhancer element (Cce). The herpes simplex virus type 1 thymidine kinase is expressed in the testes of transgenic mice under the control of a cryptic promoter. We recently reported the generation and characterization of HSV-1 DNA pol mutants [443] using a set of four overlapping cosmids and three plasmids based on the original work of Cunningham et al. Electrophoretic mobility-shift assays showed that tobacco nuclear proteins that interacted strongly with the tCUP promoter bound specifically to the 21-bp Cce element, suggesting that this sequence is probably a binding site(s) for transcription factors. The Cce element was dependent on the AT-rich element for activity indicating combinatorial control. 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 [443]. Highly reliable heterologous system for evaluating resistance of clinical herpes simplex virus isolates to nucleoside analogues.


Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.
Specific mutations in the TK gene (located between nt 186 to nt 1007 of the viral gene) were introduced into plasmid pYS-1 using the Quickchange site-directed mutagenesis kit (Stratagene, La jolla, CA). The correct nucleotide sequence of each mutated TK gene was verified by sequence analysis and compared to that of strain 17. 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. The TK and DNA pol sequences of each recombinant virus were again verified by DNA sequencing. Acyclovir susceptibility of each recombinant virus was determined by the use of a plaque reduction assay (PRA) [449]. A two-fold difference in IC50 values between the recombinant wild-type (WT) and mutant viruses was considered significant [443]. Evidence that the "active centre" of the herpes simplex virus thymidine kinase involves an interaction between three distinct regions of the polypeptide.
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 (MOI) of 2) were compared to that of the recombinant WT virus by titrating extracellular viruses collected at specific times post-infection (up to 120 h) onto Vero cells [443].Eight recombinant HSV-1 viruses (WT, TK-deleted, 4 TK mutants, 1 DNA pol mutant and 1 dual TK-DNA pol mutant), selected on the basis of their report in previous clinical or laboratory studies, were successfully generated in this study.
All recombinant mutants were resistant to ACV with the single DNA pol mutant D907V being the less resistant (4-fold 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). 2, the replication kinetics of the mutants were relatively similar to that of the WT virus up to 120 h post-infection. Our results obtained with recombinant viruses, which allows mutational studies in a homogeneous background, revealed that several mutations in conserved but also in non-conserved 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 [450]. This mutation, also described in an ACV-resistant varicella-zoster virus isolate [451], 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 [247, 253] but also in a TK-altered clinical HSV-2 isolate [287].
Of interest, the mutation P131S, located in a non-conserved region of the TK gene, also conferred an ACV-resistant phenotype. This mutation has been previously described in a laboratory-derived mutant passed in the presence of ACV [452]. Since this ACV-resistant mutant contained both the P131S TK mutation as well as 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 high level of ACV resistance whereas the single DNA pol mutation D907V, within a non-conserved gene region, induced 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. Expression of the woodchuck N-myc2 retroposon in brain and in liver tumors is driven by a cryptic N-myc promoter. The simultaneous presence of these two mutations was reported in a HSV-2 isolate from a patient with AIDS in whom ACV and foscarnet therapy sequentially failed [230].
Acyclovir-resistant herpes simplex virus infections in patients with the acquired immunodeficiency syndrome.


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 clinic, probably because this protein is not essential for viral replication in most tissues and cultured cells [233, 234]. Indeed, the replication kinetics of all tested recombinant viruses including the TK-deleted virus were similar to that of the WT virus.
In addition, mutation D907V, located in a non-conserved 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 [443].
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 to develop virtual drug phenotypes for HSV with systematic evaluation of viral mutations found in clinical specimens. Phenotypic and genotypic characterization of acyclovir-resistant herpes simplex viruses from immunocompromised patients. The yeast actin intron contains a cryptic promoter that can be switched on by preventing transcriptional interference. An ethyleneresponsive enhancer element is involved in the senescencerelated expression of the carnation glutathione-S-transferase (GSTI) gene. An altered spectrum of herpes simplex virus mutations mediated by an antimutator DNA polymerase. GUS fusion: ?-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.
A protein binding AT-rich sequence in the soybean leghemoglobin C3 promoter is a general cis element that requires proximal DNA elements to stimulate transcription. Polymorphisms of thymidine kinase gene in herpes simplex virus type 1: Analysis of clinical isolates from herpetic keratitis patients and laboratory strains. Multiple nuclear factors interact with upstream sequences of differentially regulated beta-conglycinin genes. Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants. 6th International Congress of Plant Molecular Biology, June 18–24, Quebec, Canada, Abstract S03-125. Role of genotypic analysis of the thymidine kinase gene of herpes simplex virus for determination of neurovirulence and resistance to acyclovir.
Site-directed mutagenesis of a nucleotide-binding domain in HSV-1 thymidine kinase: effects on catalytic activity. Genetic characterization of thymidine kinase from acyclovir-resistant and susceptible herpes simplex virus type 1 isolated from bone marrow transplant recipients. 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. 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. Analysis of the thymidine kinase genes from acyclovir-resistant mutants of varicella-zoster virus isolated from patients with AIDS.



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