Effects of ribonucleotide reductase inhibition on pyrimidine deoxynucleotide metabolism in acyclovir-treated cells infected with herpes simplex virus type 1.

The pyrimidine metabolism of fibroblasts infected with herpes simplex virus type 1 was studied. Herpes simplex virus type 1 infection increased the dTTP pool and thymidylate synthetase activity but reduced thymidine excretion. Addition of acyclovir to infected cells increased thymidine excretion, the dTTP pool, and thymidylate synthetase activity. Addition of a virus-specific ribonucleotide reductase inhibitor (A723U) decreased all three. The synergy between the two compounds is discussed.     

Detection of herpes simplex virus thymidine kinase polypeptides in cells labeled with 35S-methionine.

To investigate the size of herpes simplex virus (HSV) thymidine kinase (TK) polypeptides, procedures have been devised to purify the enzyme from infected cells labeled with 35S-methionine by (i) affinity chromatography on Sepharose-5'-amino-5'-deoxythymidine; (ii) preparative isoelectric focusing or preparative PAGE; and (iii) glycerol gradient centrifugation. Portions of enzyme fractions, at each purification step, were also treated with an immunoadsorbent, Sepharose-anti-HSV-1 TK immunoglobulin (IgG). Labeled polypeptides eluted from the immunoadsorbent were analyzed by electrophoresis in SDS slab gels and autoradiography. The results demonstrate that the molecular weights of HSV TK polypeptides are about 40,000. TK-negative HSV-1 mutant B2006 failed to induce the 40 K dalton polypeptide.     

Monitoring of developing graft-versus-host disease mediated by herpes simplex virus thymidine kinase gene-transduced T cells

Introduction of the HSV-Tk suicide gene into allogeneic T cells offers the possibility to control developing host-reactive cells within the context of allogeneic bone marrow transplantation (BMT). Sensitive quantitative detection methods are a prerequisite to monitor genetically modified T cells in peripheral blood and tissues to study their involvement in graft-versus-host disease (GVHD)-induced lesions as well as their disappearance or persistence after ganciclovir (GCV)-induced suicide. We monitored the alloreactivity of HSV-Tk-transduced T cells after BMT by studying their in vivo distribution and quantity in peripheral blood and in tissues in a WAG/Rij into Brown Norway fully mismatched rat allogeneic BMT model. Genetically modified T cells were quantified in blood and tissues by fluorescence-activated cell sorting, immunohistochemical analysis, and real-time quantitative polymerase chain reaction (PCR) analysis. A significant increase in the number of allogeneic HSV-Tk(+) T cells was found in particular in spleen and lymph nodes and large numbers were found in tongue, skin, and intestines. In blood, an increase in HSV-Tk(+) T cells closely preceded clinical symptoms of GVHD. Real-time quantitative PCR proved to be a fast and accurate tool by which to quantify transduced T cells both in blood and tissues. This enables the study of the in vivo alloreactivity of retrovirus-transduced cells and the response of HSV-Tk-expressing T cells to GCV-induced suicide therapy. Furthermore, we showed the potential use to study specific cause-effect relationships in a broad range of animal and clinical studies involving genetically engineered cells.     

Killing effect of the herpes simplex virus thymidine kinase/ganciclovir enzyme/prodrug system on human nasopharyngeal carcinoma cells

A promising new approach for the gene therapy of cancer is the introduction of the herpes simplex virus thymidine kinase (HSV tk) gene into tumour cells, where the HSV tk gene product converts the nontoxic prodrug ganciclovir (GCV) into its cytotoxic metabolite. We constructed a recombinant plasmid containing the HSV tk gene using standard molecular biology techniques in order to investigate whether the HSV tk/GCV enzyme/prodrug system could kill the human nasopharyngeal carcinoma cell line HNE-1. The recombinant plasmid pcDNA3.1(-) CMV. TK was transfected into the HNE-1 cells by electroporation. The expression of HSV tk by the transfected HNE-1/TK cells was confirmed by mRNA amplification and Western blotting. The growth of HNE-1/TK cells was inhibited by GCV in a dose-dependent manner. The HSV tk/GCV system also demonstrated a considerable bystander effect on co-cultured wild type HNE-1 cells. We conclude that the HSV tk/GCV system could be used as gene therapy for nasopharyngeal carcinoma.     

Assessment of a selective inhibitor of herpes simplex virus thymidine kinase (L-653,180) as therapy for experimental recurrent genital herpes.

Herpes simplex virus (HSV)-coded thymidine kinase (TK) is important in efficient reactivation of latent infection. These studies were designed to investigate whether treatment of latently infected animals with a TK inhibitor altered the natural history of recurrent HSV disease. 9-([(Z)-2-(hydroxymethyl)cyclohexyl]methyl) guanine (L-653,180) is a potent and selective nonsubstrate inhibitor of HSV TK which can suppress or delay reactivation of HSV-1 from latently infected cells in vitro without affecting viral replication. In an initial study, six female Hartley guinea pigs were treated with L-653,180 in their diet (25 mg/30 g of food) and water (300 mg/liter) for 7 days. Blood, urine, kidney, liver, spinal cord, and cerebral cortex specimens were collected. L-653,180 was detected in all specimens at concentrations which, although low, were higher than the in vitro 50% inhibitory concentration of the drug against HSV TK. In the second study, 20 female Hartley guinea pigs were randomized into two groups following recovery from primary genital HSV-2 infection. One group received L-653,180 in diet and water for 4 weeks beginning 21 days postinoculation. Animals were examined daily for recurrent lesions for 10 weeks. Treated animals experienced fewer recurrences during the treatment period but the results were not significantly different from results with controls. During the first 2-week posttreatment period, L-653,180-treated animals had significantly fewer recurrences than control animals (P = 0.02). Over the entire 10-week observation period, treated animals experienced fewer recurrences (P = 0.06). These results suggest that inhibitors of viral TK may be useful in limiting reactivation of latent virus and thus recurrent infections. In these experiments, the amount of drug that could be administered to the animals was limited by its poor solubility. Further studies with more potent and soluble inhibitors of HSV TK appear to be warranted.     

Phosphorylation of four acyclic guanosine analogs by herpes simplex virus type 2 thymidine kinase.

Four acyclic guanosine analogs, the (R) and (S) enantiomers of 9-(3,4-dihydroxybutyl)guanine, 9-(4-hydroxybutyl)guanine, and acyclovir were compared in enzyme kinetic experiments, using purified herpes simplex virus type 2 thymidine kinase. All four analogs showed competitive patterns of inhibition in the phosphorylation of thymidine by the viral thymidine kinase, but different affinities and relative rates of phosphorylation were observed.     

Specific inhibitors of herpes simplex virus thymidine kinase diminish reactivation of latent virus from explanted murine ganglia.

Two specific inhibitors of herpes simplex virus thymidine kinase, N2-phenyl-2'-deoxyguanosine and N2-(m-trifluoromethylphenyl)guanine, were tested for their ability to inhibit the reactivation of virus from explant cultures of latently infected murine trigeminal ganglia. Both compounds significantly diminished the frequency of reactivation compared with that of untreated controls.     

A sensitive method for the detection of herpes simplex virus type 2 specific thymidine kinase.

Thymidine kinase (TK) activity of uninfected and in vitro herpes simplex virus type 2 (HSV-2) infected human fetal lung cells was analyzed. Polyacrylamide gel electrophoresis was carried out, and subsequent incubation of equally divided gel slices in the presence of normal or anti-HSV-2 specific TK serum, together with different substrates, was performed. Small amounts of virus-specific enzyme could be detected using this combination of electrophoresis and antibodies and replacing ATP by CTP in the substrate medium.     

Enhanced ganciclovir killing and bystander effect of human tumor cells transduced with a retroviral vector carrying a herpes simplex virus thymidine kinase gene mutant

Gene transfer of the herpes simplex virus thymidine kinase (TK) gene associated with ganciclovir (GCV) treatment can lead to death of TK-expressing cells, and of neighboring TK- cells because of the bystander effect. Thus, a small proportion of TK+ cells in a tumor can lead to its complete regression after GCV treatment. However, a lack of efficacy of gene transfer into tumors associated with low GCV sensitivity and poor bystander effect of human cancer cells currently limit the clinical use of this suicide gene therapy approach. To increase the potency of suicide gene therapy, we have tested the GCV sensitivity and the bystander effect of TK mutants that have been previously described. After retroviral transduction of the TK mutants into human tumor cells of various origins, we have found a strong killing effect of GCV with cells expressing the mutants TK30 or TKF161C. The GCV sensitivity of several human tumor cell types expressing TK30 was 9- to 500-fold higher than cells containing wild-type TK. Furthermore, TK30-expressing cells were able to kill bystander cells much more efficiently than TK-expressing cells. Thus, TK30 mutant is a promising candidate for suicide gene therapy clinical trials.     

Influence of acyclovir and bucyclovir on nucleotide pools in cells infected with herpes simplex virus type 1.

The effects of the acyclic guanosine analogs acyclovir (ACV) and (R)-9-(3,4-dihydroxybutyl)guanine (bucyclovir, BCV) on the deoxyribonucleoside triphosphate (dNTP) pools of herpes simplex type 1 (HSV-1)-infected African green monkey kidney (GMK) and human embryonic lung fibroblast (HL) cells were investigated. HSV-1 infection increased the dNTP pools in both cell types compared with those in uninfected cells. Mock-infected GMK cells showed a 10-fold-higher dTTP concentration than comparable HL cells. ACV or BCV treatment of HSV-1-infected cells yielded further increases of the dNTP pools. ACV- or BCV-treated, HSV-1-infected HL cells showed 20- to 50-fold-higher concentrations of ACV triphosphate and BCV triphosphate, respectively, than similarly treated GMK cells. This is in accord with previous results, which showed that ACV and BCV are less active in GMK cells than in HL cells. This difference in activity is attributed to the substantial deoxythymidine pools previously found in GMK cells. The results are discussed in relation to known metabolic and kinetic parameters.     

Localization of the thymidine kinase gene of herpes simplex virus type 2 (333)

The thymidine kinase (tk) gene of herpes simplex virus type 2 (HSV-2) has been identified on purified restriction endonuclease fragments of HSV-2 DNA. These fragments were localized on the physical map of HSV-2 DNA. A detailed map of the HSV-2 tk gene region has been constructed which locates the tk gene between 0.285 and 0.310 map units [3.8 kilobase pairs (kb)], whereby tk gene sequences are at least present between 0.299 and 0.303 map units (0.6 kb). Mouse tk- cells that were biochemically transformed contain HSV-2 tk gene DNA sequences that are integrated in the cellular genome.     

Clinical isolate of herpes simplex virus type 2 that induces a thymidine kinase with altered substrate specificity

In vitro and in vivo studies were done on a herpes simplex virus type 2 strain recovered from a patient on acyclovir (ACV) which was ACV resistant but expressed thymidine (dThd) kinase (EC 2.7.1.21) activity. Plaque-purified clones derived from the original clinical sample were heterogeneous with respect to plaque size and drug susceptibility. The heterogeneity of this viral mixture was also evident from varied 125I-labeled 5-iodo-2'-deoxycytidine autoradiographic patterns and from varied expression of dThd kinase-associated phosphorylating activities. Four clones from this mixture were 1-beta-D-arabinofuranosylthymine (ara-T) susceptible and ACV resistant. Extracts of cells infected with these clones catalyzed the phosphorylation of ara-T but little of ACV. The virus-coded dThd kinase was purified from one of these clones to determine whether its substrate specificity was altered. The amount of virus-coded dThd phosphorylating activity with the cell extracts was estimated to be sevenfold lower with the resistant clone than with the MS strain of herpes simplex virus type 2. The dThd kinase eluted from a dThd-agarose affinity column under the same conditions with extracts from both sources and substrate saturations of both enzymes by acyclic nucleoside analog phosphate acceptors were classical hyperbolic functions. However, there were significant differences in the kinetic parameters of substrates between the two enzymes. Apparent Km (Km') values for dThd, deoxycytidine, ara-T, ACV, and the acyclic guanosine analog 9-[[2-hydroxyl-1-(hydroxymethyl)ethoxy]methyl]guaine (BW B759U) were 2- to 60-fold higher with the variant enzyme than with the enzyme from laboratory strain MS. Comparing these two enzymes, relative maximal phosphorylation rates (Vm) were eightfold lower for ACV but unchanged for BW B759U. In contrast, the relative rates for deoxycytidine and ara-T were eight- and twofold higher, respectively. The surprisingly good substrate activity with BW B759U compared with that of ACV (Vm/Km' = 0.39 versus 0.01) coincided with susceptibility of the ACV-resistant virus to BW B759U. This clinical variant retained its pathogenicity for mice and was only moderately less neurovirulent than wild-type virus. Although such mutants have the potential to induce illness less responsive to therapy, the recurrence from which the isolate was obtained was typical for this patient in severity and duration. Since this episode, the patient has been treated successfully with ACV.     

单纯疱疹病毒胸苷激酶基因重组真核表达载体的构建及在烫伤大鼠皮肤组织中的表达

目的：构建单纯疱疹病毒胸苷激酶（HSV—tk）基因重组真核表达载体pcDNA3．1／HSV—tk，并观察其在烫伤大鼠皮肤中的表达。方法：实验于2003～07／12在解放军第一军医大学热带卫生学系实验室进行。在原核表达质粒pUChHyTk基础上，利用分子克隆技术，构建重组真核表达质粒pcDNA3．1／HSV—tk。取20只Wistar大鼠，全麻后背部浸入95℃水浴锅中，烫伤12s，造成30％Ⅲ度烫伤，烫伤后随机分为两组（n=10）：①pcDNA3．1／tk组：烫伤后当天及第1，2，3，4周后于大鼠左后背部创面边缘（距创面边缘0．5cm）皮下注射脂质体和重组质粒的混合物192μL[3．6μg pcDNA3．1／tk（2μL）＋10μL脂质体＋180μL生理盐水】。②pcDNA3．1组：注射时间和方法同前，脂质体和质粒混合物为3．0μg pcDNA3．1（2μL）＋10μL脂质体＋180μL生理盐水。伤后5周断头处死大鼠，取注射点附近皮肤，用反转录-聚合酶链反应法检测HSV—tk基因的表达。结果：20只大鼠全部进入结果分析。大鼠重组质粒经酶切鉴定与预期结果一致，DNA测序结果表明tk基因已正确插入到pcDNA3．1质粒，说明了重组真核表达载体pcDNA3.1／HSV—tk已成功构建。反转录-聚合酶链反应结果显示脂质体介导的HSV—tk基因转移可在创面成纤维细胞中出现阳性表达。结论：应用分子克隆技术成功地构建了重组真核表达载体pcDNA3．1／HSV—tk，并在烫伤大鼠皮肤中得到了阳性表达。     

单纯疱疹病毒胸苷激酶基因重组真核表达载体的构建及在烫伤大鼠皮肤组织中的表达

目的:构建单纯疱疹病毒胸苷激酶(HSV-tk)基因重组真核表达载体pcDNA3.1/HSV-tk,并观察其在烫伤大鼠皮肤中的表达。方法:实验于2003-07/12在解放军第一军医大学热带卫生学系实验室进行。在原核表达质粒pUChHyTk基础上,利用分子克隆技术,构建重组真核表达质粒pcDNA3.1/HSV-tk。取20只Wistar大鼠,全麻后背部浸入95℃水浴锅中,烫伤12s,造成30%Ⅲ度烫伤,烫伤后随机分为两组(n=10):①pcDNA3.1/tk组:烫伤后当天及第1,2,3,4周后于大鼠左后背部创面边缘(距创面边缘0.5cm)皮下注射脂质体和重组质粒的混合物192μL眼3.6μgpcDNA3.1/tk(2μL) 10μL脂质体 180μL生理盐水演。②pcDNA3.1组:注射时间和方法同前,脂质体和质粒混合物为3.0μgpcDNA3.1(2μL) 10μL脂质体 180μL生理盐水。伤后5周断头处死大鼠,取注射点附近皮肤,用反转录-聚合酶链反应法检测HSV-tk基因的表达。结果:20只大鼠全部进入结果分析。大鼠重组质粒经酶切鉴定与预期结果一致,DNA测序结果表明tk基因已正确插入到pcDNA3.1质粒,说明了重组真核表达载体pcDNA3.1/HSV-tk已成功构建。反转录-聚合酶链反应结果显示脂质体介导的HSV-tk基因转移可在创面成纤维细胞中出现阳性表达。结论:应用分子克隆技术成功地构建了重组真核表达载体pcDNA3.1/HSV-tk,并在烫伤大鼠皮肤中得到了阳性表达。     

DNA-binding proteins of cells infected by herpes simplex virus type 1 and type 2.

Proteins showing affinity for DNA in HSV-1-and HSV-2-infected cells were compared by DNA-cellulose chromatography and PAGE. The proteins observed depended on the type of virus used to infect the cell; however, several examples of analogous polypeptides were present in cells infected by both virus types. Proteins showing highest affinity for DNA-cellulose were similar in molecular size in cells infected by both virus types.     

Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22.

We demonstrate that fusion proteins consisting of the herpes simplex virus (HSV) transport protein VP22 linked in frame to HSV thymidine kinase (tk) retain the ability to be transported between cells. In vivo radiolabelling experiments and in vitro assays show that the fusion proteins also retain tk activity. When transfected COS cells, acting as a source of the VP22-tk chimera, were co-plated on to gap junction-negative neuroblastoma cells, ganciclovir treatment induced efficient cell death in the recipient neuroblastoma cell monolayer. No such effect was observed with COS cells transfected with tk alone. Tumours established in mice with neuroblastoma cell lines expressing VP22-tk regressed upon administration of ganciclovir. Furthermore tumours established from 50:50 mixtures of VP22-tk transduced and nontransduced cells also regressed while no significant effect was observed in similar experiments with cells transduced with tk alone. VP22 mediated transport may thus have application in a clinical setting to amplify delivery of the target protein in enzyme-prodrug protocols.