Mechanism of resistance to acyclovir in thymidine kinase mutants from Herpes simplex virus type 1: a computational approach

Abstract

Herpes simplex virus type 1 (HSV-1) infections affect about two-thirds of the world population, and the standard treatment consists of acyclovir (ACV) and its analogs, which interact with thymidine kinase (TK) blocking viral replication. Lately, the emergence of ACV-resistant strains has been reported, especially associated with TK mutations. In this context, ACV therapy fails against isolates encoding Y172C and Y53H/R163H TK mutants, but the molecular mechanism of drug resistance remains unclear. Thus, we examined the effects of these mutations on ACV and the cofactor ATP binding through molecular modeling approaches. We showed that Y172C prevents the anchoring of the aromatic ring of ACV through π–π stacking interactions, leading to an inversed binding mode and different interactions. On the other hand, Y53H/R163H remarkably affected the cofactor binding mode which shifted away from its binding site and also influenced the interaction network of ACV. This is likely due to the loss of polar interactions with R163 residue. Unlike what was observed in the wild-type complex, both drug and cofactor binding poses were not well positioned to allow the phosphorylation reaction which explains the resistance observed. Moreover, energy analysis corroborated the experimental data and showed lower theoretical affinity of ACV with mutant enzymes resulted from energetic loss in polar solvation in Y172C and electrostatic terms in Y53H/R163H mutant enzyme. Therefore, our study shed light on the resistance mechanism toward ACV of two mutant TKs identified in clinical HSV-1 strains and may further support the development of new anti-herpetic drugs to treat resistant infections.

Communicated by Ramaswamy H. Sarma     

Replication-competent, oncolytic herpes simplex virus type 1 mutants induce a bystander effect following ganciclovir treatment

Cells expressing herpes simplex virus (HSV) thymidine kinase (tk) are killed by ganciclovir (GCV). Adjacent cells without HSV-tk also die, a phenomenon known as the 'bystander effect'. However, there is no evidence that replication-competent HSV induces a bystander effect in the presence of GCV. Therefore, we investigated the bystander effect in HEp-2 cells infected with replication-competent, oncolytic HSV-1 mutants, hrR3 and HF10. In cells infected at a multiplicity of infection (MOI) of 3, GCV did not induce apoptosis. At low MOIs of 0.3 and 0.03, however, a number of adjacent, uninfected cells apoptosed following GCV treatment. Irrespective of GCV treatment, HEp-2 cells expressed minimal levels of connexin 43 (Cx43). However, Cx43 expression was enhanced by GCV in response to infection with HF10 at an MOI of 0.3, but not at an MOI of 3. Expression of other proteins involved in gap junctions, including Cx26 and Cx40, was not augmented under these conditions. The PKA and PI3K signal transduction pathways are likely involved in enhanced Cx43 expression as inhibitors of these pathways prevented Cx43 upregulation. These results suggest that infection with replication-competent HSV-1 induces the bystander effect in cells treated with GCV because of efficient intercellular transport of active GCV through abundant gap junctions.     

A new N-methyl thymine derivative comprising a dihydroxyisobutenyl unit as ligand for thymidine kinase of herpes simplex virus type 1 (HSV-1 TK)

Molecular modeling and phosphorylation assay in vitro were employed to select a novel unsaturated 1,3-dihydroxyisobutenyl thymine derivative 6 as ligand for HSV-1 TK which may be of interest as lead for the development of an positron emission tomography (PET) imaging agent. Compound 6 was successfully prepared using modified approaches. A significant improvement over the syntheses involving pathways A and B (1% and 3% overall yield, respectively), was observed using synthetic route C (14% overall yield).     

The effect of substrate binding on the conformation and structural stability of Herpes simplex virus type 1 thymidine kinase

The structure of Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) is known at high resolution in complex with a series of ligands and exhibits important structural similarities to the nucleoside monophosphate (NMP) kinase family, which are known to show large conformational changes upon binding of substrates. The effect of substrate binding on the conformation and structural stability of TK(HSV1), measured by thermal denaturation experiments, far-UV circular dichroism (CD) and fluorescence is described, and the results indicate that the conformation of the ligand-free TK(HSV1) is less ordered and less stable compared to the ligated enzyme. Furthermore, two crystal structures of TK(HSV1) in complex with two new ligands, HPT and HMTT, refined to 2.2 A are presented. Although TK(HSV1):HPT does not exhibit any significant deviations from the model of TK(HSV1):dT, the TK(HSV1):HMTT complex displays a unique conformationally altered active site resulting in a lowered thermal stability of this complex. Moreover, we show that binding affinity and binding mode of the ligand correlate with thermal stability of the complex. We use this correlation to propose a method to estimate binding constants for new TK(HSV1)substrates using thermal denaturation measurements monitored by CD spectroscopy. The kinetic and structural results of both test substrates HPT and HMTT show that the CD thermal denaturation system is very sensitive to conformational changes caused by unusual binding of a substrate analog.     

2,3-Dihydroxy-quinoxaline induces ATPase activity of Herpes Simplex Virus thymidine kinase

2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with K_i = 250 μM (95% CI: 106–405 μM) and dose-dependently increased the rate of the ATP hydrolysis with K_M = 112 μM (95% CI: 28–195 μM). The kinetic scheme consistent with this experimental data is proposed.     

Mutation spectra of herpes simplex virus type 1 thymidine kinase mutants

To examine whether the exonuclease activity intrinsic to the polymerase (Pol) of herpes simplex virus type 1 can influence the mutational spectra, we applied the denaturing gradient gel electrophoresis (DGGE) system combined with sequencing to characterize thymidine kinase mutants isolated from both the wild-type virus and a mutant deficient in exonuclease activity, Y7. Wild-type viruses produced predominantly frameshift mutations (67%), whereas Y7 replicated a significantly lower proportion of frameshifts (21%; P < 0.005). Furthermore, the majority of substitutions were transitional changes in both groups, although they distributed differently. The implications of these findings are discussed.     

溶瘤I型单纯疱疹病毒的研究进展

溶瘤病毒(Oncolytic virus,OV)是可以靶向感染并杀伤肿瘤细胞的一类病毒,其中溶瘤I型单纯疱疹病毒(Oncolytic herpes simplex virus type 1,OHSV-1)是目前研究最多的溶瘤病毒之一,可通过多种策略进行构建,已有多种OHSV-1进入临床试验,大量结果显示其具有较好的安全性和有效性。本文主要介绍OHSV-1的分子生物学特性与优势、主要的开发及靶向性策略、各类OHSV-1的研究进展以及目前存在的问题等。     

Variable expression of the herpes simplex virus thymidine kinase gene in Nicotiana tabacum affects negative selection

The potentials and limitations of negative-selection systems based on the human herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which causes sensitivity to the nucleoside analog ganciclovir, were examined in tobacco as a model system. There were great differences between individual HSVtk⁺ transgenic plants in ganciclovir sensitivity. Inhibition of growth while under selection correlated with HSVtk-tianscnpt levels. Negative selection against HSVtk⁺ transformants at the level of Agrobacterium-mediated transformation using a ganciclo-vir/kanamycin double-selection medium (the positive selection marker neomycin phosphotransferase-II gene was in the transformation vector) resulted in a three- to six-fold reduction in the frequency of kanamycin-resistant shoots. The efficiency of negative selection in this case was limited due to the great variation in HSVtk expression, i.e., the frequently occurring transformants with low, or no, ganciclovir sensitivity escaping negative selection. Two independently constructed HSVtk genes showed the same variability of the phenotype in Nicotiana tabacum transformants. Distinct phenotypes, ranging from no regeneration through abnormal or delayed regeneration, were observed when leaf segments were placed on shoot-inducing medium supplemented with 10^–6–10^–3 M ganciclovir. The highest HSVtk mRNA and ganciclovir sensitivity levels were observed in plants which were transformed with the pSLJ882 chimeric construct. The pSLJ882 plant expression vector carried the coding sequence of HSVtk, whereas plasmid pCX305.1 carried an HSVtk construct retaining the untranslated 5 leader and viral 3 regions. The pCX305.1 transformants showed, at most, a delayed formation of shoots with thin stems and very narrow leaves. Ganciclovir sensitivity showed typical Mendelian segregation. A gene-dosage effect was also seen at the seedling level in the progeny of two transgenic lines.     

Characterization of herpes simplex virus type 1 thymidine kinase mutants selected under a single round of high-dose brivudin

A broad variety of herpes simplex virus type 1 clones was selected under a single round of high-dose selection with brivudin. Mutations in the thymidine kinase (TK) genes consisted of 42% frameshift mutations within homopolymer repeats of G's and C's and single nucleotide substitutions (58%) that produced stop codons (Q261 and R281) or a new codon at the site of the substitution (A168T, R51W, G59W, G206R, R220H, Y239S, and T287 M). The A168T change, associated with an altered TK phenotype, proved to be the most commonly selected substitution. For the different mutants, a correlation between phenotype, genotype, and in vivo neurovirulence was observed.     

Differential ganciclovir-mediated cell killing by glutamine 125 mutants of herpes simplex virus type 1 thymidine kinase

The therapeutic combination of the herpesvirus simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and the prodrug, ganciclovir (GCV), has found great utility for the treatment of many types of cancer. After initial phosphorylation of GCV by HSV-1 TK, cellular kinases generate the toxic GCV-triphosphate metabolite that is incorporated into DNA and eventually leads to tumor cell death. The cellular and pharmacological mechanisms by which metabolites of GCV lead to cell death are still poorly defined. To begin to address these mechanisms, different mutated forms of HSV-1 TK at residue Gln-125 that have distinct substrate properties were expressed in mammalian cell lines. It was found that expression of the Asn-125 HSV-1 TK mutant in two cell lines, NIH3T3 and HCT-116, was equally effective as wild-type HSV-1 TK for metabolism and sensitivity to GCV, bystander effect killing and induction of apoptosis. The major difference between the two enzymes was the lack of deoxypyrimidine metabolism in the Asn-125 TK-expressing cells. In HCT-116 cells expressing the Glu-125 TK mutant, GCV metabolism was greatly attenuated, yet at higher GCV concentrations, cell sensitivity to the drug and bystander effect killing were diminished but still effective. Cell cycle analysis, 4', 6'-diamidine-2'-phenylindoledihydrochloride staining, and caspase 3 activation assays indicated different cell death responses in the Glu-125 TK-expressing cells as compared with the wild-type HSV-1 TK or Asn-125 TK-expressing cells. A mechanistic hypothesis to explain these results based on the differences in GCV-triphosphate metabolite levels is presented.     

Characterization of multiple nuclear localization signals in herpes simplex virus type 1 thymidine kinase.

We have reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) fused with green fluorescent protein (GFP) is localized in the nucleus of HSV-1 TK-GFP gene-transfected cells (Degrève et al. (1998) J. Virol. 72, 9535-9543). Deletion of the N-terminal 34 amino acids or selective mutation of the nonapeptide (25)RRTALRPRR(33), located in the N-terminal region of HSV-1 TK, resulted in the loss of the specific nuclear localization of HSV-1 TK. Utilizing information on the crystallographic structure of HSV-1 TK, we have now identified three additional putative nuclear localization signals and evaluated their potential role in the nuclear trafficking of HSV-1 TK by site-directed mutagenesis. We found that the sites containing the amino acids R236-R237 and K317-R318 are absolutely required for specific nuclear targeting of HSV-1 TK. The K317-R318 region, located at the interface between the two monomers in the dimeric HSV-1 TK structure, could act as a nuclear localization signal for monomeric HSV-1 TK. Alternatively, crystallographic data indicate that R318 might be essential for the formation of the TK dimer, and therefore it is required if HSV-1 TK is transported as a dimer.     

Synthesis of the herpes simplex virus type 1 trans-activator Vmw65 in insect cells using a baculovirus vector

Vmw65, the Herpes Simplex Virus trans-activator of immediate-early genes, was expressed in insect cells using a recombinant baculovirus expression vector and partially purified. Insect cell-derived Vmw65 was shown to be indistinguishable from authentic Vmw65 present in purified HSV-1 virions based on electrophoretic mobility, immunoreactivity with a monoclonal antibody, and ability to interact with cellular factors to form a protein/DNA complex with oligonucleotides containing a TAATGARAT element.Abbreviations AcNPV Autographica californica nuclear polyhedrosis virus - HSV Herpes Simplex Virus - IE Immediate Early - moi multiplicity of infection - Sf9 Spodoptera frugiperda cells     

Characterization of oligosaccharides of highly purified glycoprotein gC of herpes simplex virus type 1 (HSV-1)

The envelope membrane glycoprotein gC of HSV-1 was purified from Triton X-100 extracts of virus-infected BHK-21 or HEp-2 cells by a single step immuno-affinity column using monoclonal anti-gC antibody. The analysis of the purified [³H]G1cN labeled glycoprotein gC (by gel filtration on Bio-Gel P4) before and after digestion with endo-β-N-acetylglucosaminidase (endo D) indicated that gC contains Asn-linked “complex type” oligosaccharides. No “high mannose” type oligosaccharides were detected. Fractionation of radio-labeled glycopeptides of gC on a column of concanavalin A-sepharose suggested that glycopeptides have “diantennary” and “triantennary” and/or “tetra antennary” structures. Tunicamycin inhibited the incorporation of [¹⁴C]GalN or [³H]GlcN into gC in HSV-1 infected BHK-21 or HEp-2 cells. Gel filtration analysis of [³H]GlcN labeled gC following β-elimination reaction failed to indicate O-glycosidically linked oligosaccharides.     

Co-expression of bfl-1 enhances host response in the herpes simplex virus-thymidine kinase/ganciclovir gene therapy system

Anticancer suicide gene therapy using herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir (GCV) features the unique advantage of being able to elicit brisk host immune response against tumors and the host response reportedly can be potentiated with the co-expression of other appropriate immune- or apoptosis-related genes. We introduced a novel antiapoptotic gene, bfl-1, to test its applicability in the HSV-tk/GCV system. CT-26 murine colon cancer cells transfected with HSV-tk, alone or in combination with bcl-xL or bfl-1, were either grown in vitro or injected into syngeneic mice, followed by GCV administration. The co-expression of bfl-1 was associated with the upregulation of CD95 and CD40 ligand (CD40L) in vitro and with pronounced intratumoral T-lymphocyte infiltration in vivo. These results add to the previous findings that antiapoptotic genes can be used as an adjunctive component in the HSV-tk/GCV system to enhance host immune response against tumors.