Single nucleotide polymorphisms of thymidine kinase and DNA polymerase genes in clinical herpes simplex virus type 1 isolates associated with different resistance phenotypes

cHerpes simplex virus 1 (HSV-1) and HSV-2 remain major human pathogens despite the development of anti-HSV therapeutics as some of the first antiviral drugs. Current therapies are incompletely effective and frequently drive the evolution of drug-resistant mutants. We recently determined that certain natural troponoid compounds such as ␤-thujaplicinol readily suppress HSV-1 and HSV-2 replication. Here, we screened 26 synthetic ␣-hydroxytropolones with the goals of determining a preliminary structure-activity relationship for the ␣-hydroxytropolone pharmacophore and providing a starting point for future optimization studies. Twenty-five compounds inhibited HSV-1 and HSV-2 replication at 50 M, and 10 compounds inhibited HSV-1 and HSV-2 at 5 M, with similar inhibition patterns and potencies against both viruses being observed. The two most powerful inhibitors shared a common biphenyl side chain, were capable of inhibiting HSV-1 and HSV-2 with a 50% effective concentration (EC 50 ) of 81 to 210 nM, and also strongly inhibited acyclovir-resistant mutants. Moderate to low cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC 50 ] of 50 to >100 M). Therapeutic indexes ranged from >170 to >1,200. These data indicate that troponoids and specifically ␣-hydroxytropolones are a promising lead scaffold for development as anti-HSV drugs provided that toxicity can be further minimized. Troponoid drugs are envisioned to be employed alone or in combination with existing nucleos(t)ide analogs to suppress HSV replication far enough to prevent viral shedding and to limit the development of or treat nucleos(t)ide analog-resistant mutants.

Section: Discussionmentioning

confidence: 99%

Synthetic α-Hydroxytropolones Inhibit Replication of Wild-Type and Acyclovir-Resistant Herpes Simplex Viruses

Ireland

Tavis

D’Erasmo

et al. 2016

“…Herpes simplex virus 1 was identified and HSV-2 could be excluded by diagnostic PCR as described previously (21)(22)(23). All HSV-1 strains were isolated and propagated in African green monkey kidney Vero 76 cells (ATCC, CRL 1587) and/or human embryonic lung fibroblasts (HELFs).…”

Section: Methodsmentioning

confidence: 99%

“…Only isolates with novel or unclear nonsynonymous mutations in the DNA Pol or ACV resistance-associated genotype were tested against FOS (AstraZeneca, Wilmslow, United Kingdom). All phenotypic analyses were performed using a plaque reduction assay, including the formazan test according to the method described previously (16,17,19,21). In short, Vero 76 cells, only used for testing FOS, or human Caucasian fetal lung fibroblasts of the cell line Wi 38 (European Collection of Cell Cultures, Salisbury, United Kingdom), exclusively used for analyzing the phenotype to ACV, were seeded at a density of 10 5 ml Ϫ1 .…”

Section: Methodsmentioning

confidence: 99%

“…Usually, phenotypic HSV-1 assays allow clear interpretation of novel mutations causing drug resistance, but in clinical practice, time-con-suming phenotypic testing delays resistance-adjusted treatment. Since there are no equivalent methods for resistance screening that can be used in clinical diagnostics, the comparison of genotype and phenotype is still needed to characterize the resistance association of unknown amino acid changes (21).…”

mentioning

confidence: 99%

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Sequence Analysis of Herpes Simplex Virus 1 Thymidine Kinase and DNA Polymerase Genes from over 300 Clinical Isolates from 1973 to 2014 Finds Novel Mutations That May Be Relevant for Development of Antiviral Resistance

Schmidt

Bohn-Wippert

Zell

et al. 2015

Self Cite

A total of 302 clinical herpes simplex virus 1 (HSV-1) strains, collected over 4 decades from 1973 to 2014, were characterized retrospectively for drug resistance. All HSV-1 isolates were analyzed genotypically for nonsynonymous mutations in the thymidine kinase (TK) and DNA polymerase (Pol) genes. The resistance phenotype against acyclovir (ACV) and/or foscarnet (FOS) was examined in the case of novel, unclear, or resistance-related mutations. Twenty-six novel natural polymorphisms could be detected in the TK gene and 69 in the DNA Pol gene. Furthermore, three novel resistance-associated mutations (two in the TK gene and one in the DNA Pol gene) were analyzed, and eight known but hitherto unclear amino acid substitutions (two encoded in TK and six in the DNA Pol gene) could be clarified. Between 1973 and 2014, the distribution of amino acid changes related to the natural gene polymorphisms of TK and DNA Pol remained largely stable. Resistance to ACV was confirmed phenotypically for 16 isolates, and resistance to ACV plus FOS was confirmed for 1 isolate. Acyclovir-resistant strains were observed from the year 1995 onwards, predominantly in immunosuppressed patients, especially those with stem cell transplantation, and the number of ACV-resistant strains increased during the last 2 decades. The data confirm the strong genetic variability among HIV-1 isolates, which is more pronounced in the DNA Pol gene than in the TK gene, and will facilitate considerably the rapid genotypic diagnosis of HSV-1 resistance. Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen throughout the world. After primary infection occurring mostly during infancy, the virus remains latently for life in sensory local ganglia. Thereafter, HSV-1 may be reactivated and can cause usually self-limiting orolabial lesions in immunocompetent individuals. In contrast, immunocompromised patients often develop chronic diseases associated with painful and widespread exanthema and/or enanthema. Since its marketing in the 1980s, acyclovir (ACV), a guanosine analog, has been the drug of choice for the treatment of HSV infections; its active form is ACV triphosphate (1, 2). Foscarnet (FOS), a pyrophosphate analog, directly inhibits the viral DNA polymerase (Pol) and can be used successfully as an alternative drug in case of ACV resistance.Acyclovir-resistant HSV-1 isolates have a remarkable clinical prevalence, especially in immunocompromised patients. Summarizing published papers of recent years, the prevalence of ACVresistant HSV strains in immunosuppressed patients ranges between 2.5% and 10.9% (3, 4). For immunocompetent patients, studies between 1985 and 1993 showed that ACV resistance in clinical HSV isolates is Ͻ1.0%, very low (5-8). An overall increase in ACV-resistant HSV strains could not be established in a subsequent study (9).The HSV thymidine kinase (TK) protein is responsible for the conversion of ACV to its active form, and 95% of the mutations encoding antiviral resistance are found in the TK gene (10). Only a few cases of resistanc...

“…The cells were infected with a multiplicity of infection of 0.12. The antivirals were applied at a final half-log dilution over a previously described range (26). After incubation for 3 days, viral plaques were counted under a fluorescence microscope (Diaphot; Nikon, Japan).…”

Section: Methodsmentioning

confidence: 99%

Drug Resistance of Clinical Varicella-Zoster Virus Strains Confirmed by Recombinant Thymidine Kinase Expression and by Targeted Resistance Mutagenesis of a Cloned Wild-Type Isolate

Brunnemann

Bohn-Wippert

Zell

et al. 2015

Self Cite

In this study, approaches were developed to examine the phenotypes of nonviable clinical varicella-zoster virus (VZV) strains with amino acid substitutions in the thymidine kinase (TK) (open reading frame 36 [ORF36]) and/or DNA polymerase (Pol) (ORF28) suspected to cause resistance to antivirals. Initially, recombinant TK proteins containing amino acid substitutions described as known or suspected causes of antiviral resistance were analyzed by measuring the TK activity by applying a modified commercial enzyme immunoassay. To examine the effects of these TK and Pol substitutions on the replication of recombinant virus strains, the method of en passant mutagenesis was used. Targeted mutations within ORF36 and/or ORF28 and an autonomously expressed gene of the monomeric red fluorescent protein for plaque identification were introduced into the European wild-type VZV strain HJO. Plaque reduction assays revealed that the amino acid substitutions with unknown functions in TK, Q303stop, N334stop, A163stop, and the deletion of amino acids 7 to 74 aa (⌬aa 7 to 74), were associated with resistance against acyclovir (ACV), penciclovir, or brivudine, whereas the L73I substitution and the Pol substitutions T237K and A955T revealed sensitive viral phenotypes. The results were confirmed by quantitative PCR by measuring the viral load under increasing ACV concentrations. In conclusion, analyzing the enzymatic activities of recombinant TK proteins represent a useful tool for evaluating the significance of amino acid substitutions in the antiviral resistance of clinical VZV strains. However, direct testing of replication-competent viruses by the introduction of nonsynonymous mutations in a VZV bacterial artificial chromosome using en passant mutagenesis led to reliable phenotypic characterization results. H erpes zoster is caused by endogenous varicella-zoster virus (VZV) reactivation, especially in the elderly or in patients with immunodeficiencies. The disease occurs in 5 to 32% of transplant patients (1) and is related to significant morbidity and mortality, with fatality rates of up to 28% (1-3). The efficacy of antiviral therapy was demonstrated by multiple randomized controlled studies. Acyclovir (ACV), its prodrug valacyclovir, famciclovir (the prodrug of penciclovir [PCV]), and (E)-5-(2-bromovinyl)-2=-deoxyuridine (brivudine [BVDU]) are approved as therapeutics in many countries to treat VZV (4). These nucleoside analogues are phosphorylated by the viral thymidine kinase (TK) (open reading frame 36 [ORF36]) and cellular kinases to form a triphosphate that blocks viral DNA polymerase (Pol) (ORF28) by acting as competitive inhibitors and/or DNA chain terminators. Acyclovir resistance has been reported in immunocompromised, but not in immunocompetent, patients (5). If a patient shows therapeutic failure within 7 to 10 days, drug susceptibility should be evaluated (4, 6, 7), and alternative drugs are required. Foscarnet (FOS) and cidofovir (CDV), both inhibitors of Pol (8, 9), act independently of the viral TK and are recomm...