Location and cloning of the herpes simplex virus type 2 thymidine kinase gene.

The herpes simplex virus type 2 thymidine kinase gene has been mapped to a position colinear with the herpes simplex virus type 1 thymidine kinase gene and cloned within a 4.0-kilobase fragment in pBR 322.     

Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.     

Properties of a novel thymidine kinase induced by an acyclovir-resistant herpes simplex virus type 1 mutant.

The acyclovir-resistant mutant of herpes simplex virus type 1, SC16 S1, induced reduced levels of thymidine kinase activity (ca. 25% reduction) in infected cells. The activity appeared with kinetics similar to that in wild type-infected cells, and pulse-labeling experiments showed that the thymidine kinase polypeptide was synthesized at a similar rate. We showed that the enzyme was virus specific by inactivating it with antiserum raised against herpes simplex virus-infected cell proteins. The enzyme induced by the mutant had reduced electrophoretic mobility in nondenaturing gels, decreased thermal stability, and decreased affinity for several different substrates (assessed by measurement of Km values) compared with the enzyme induced by the wild type. From the data obtained we conclude that the thymidine kinase induced by the mutant has an altered specificity, probably resulting from an amino acid substitution which affects the primary binding site for nucleosides and nucleoside analogs.     

Molecular modeling of the binding of 5-substituted 2'-deoxyuridine substrates to thymidine kinase of herpes simplex virus type-1

In an earlier study, De Winter and Herdewijn (J. Med. Chem. 1996, 37, 4727-4737) studied the binding of various 5-substituted 2'-deoxyuridine substrates to thymidine kinase of herpes simplex virus type-1. They used a computational procedure that achieves good correlation with experimentally determined IC50 values. We applied an alternative procedure to the same deoxyuridine substrates, using only three readily calculated quantities-the binding energy, the molecular surface area, and a flexibility factor. Our simplified method achieves the same degree of correlation with the IC50 values as did the earlier procedure. We then applied this procedure to examine the binding of various 5-substituted pyrimidine 1,5-anhydrohexitol substrates to thymidine kinase.     

Encapsidation and expression of the herpes thymidine kinase gene in polyoma virus.

A recombinant DNA of 5,150 base pairs was prepared containing the intact early region of polyoma virus, including the viral origin of replication and the structural sequences of the herpes simplex virus type 1 thymidine kinase gene. Although no thymidine kinase activity was detected when herpes structural sequences alone were transfected into cells, activity was produced when the structural gene followed the polyoma early region. The recombinant DNA was encapsidated into polyoma virions when cotransfected into mouse 3T6 cells with helper DNA from an early polyoma virus mutant. Herpes thymidine kinase activity was detected by a rapid in situ autoradiographic assay in which [125]iododeoxycytidine was utilized as a substrate for the viral but not the cellular enzyme.     

The herpes simplex virus thymidine kinase gene is not transcribed in Saccharomyces cerevisiae

The herpes simplex virus thymidine kinase gene has been cloned into a chimeric yeast plasmid cloning vehicle and transformed into appropriate yeast strains. Plasmids carrying the herpes simplex virus thymidine kinase gene can be propagated as autonomously replicating plasmids, but no RNA specific to the thymidine kinase coding sequence was detected.     

Design, synthesis and enzymatic activity of highly selective human mitochondrial thymidine kinase inhibitors

Highly selective arabinofuranosyl nucleosides, which inhibit the mitochondrial thymidine kinase (TK-2) without affecting the closely related herpes simplex virus type 1 thymidine kinase (HSV-1 TK), varicella-zoster virus thymidine kinase (VZV-TK), cytosolic thymidine kinase (TK-1) or the multifunctional Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK), have been obtained. SAR studies indicate a close relation between the length of the substituent at the 2' position of the arabinofuranosyl moiety and the inhibitory activity.     

Development and validation of a capillary electrophoresis method for the characterization of herpes simplex virus type 1 (HSV-1) thymidine kinase substrates and inhibitors

A fast, convenient capillary electrophoresis (CE) method was developed for monitoring the enzymatic reaction of herpes simplex virus type 1 thymidine kinase (HSV-1 TK). The reaction was performed in a test tube followed by quantitative analysis of the products. The optimized CE conditions were as follows: polyacrylamide-coated capillary (20 cm effective length x 50 microm), electrokinetic injection for 30s, 50 mM phosphate buffer at pH 6.5, constant current of -60 microA, UV detection at 210 nm, UMP or cAMP were used as internal standards. Phosphorylated products eluted within less than 7 min. The limits of detection were 0.36 microM for dTMP and 0.86 microM for GMP. The method was used to study enzyme kinetics, and to investigate alternative substrates and inhibitors.     

The rational of catalytic activity of herpes simplex virus thymidine kinase. a combined biochemical and quantum chemical study

Most antiherpes therapies exploit the large substrate acceptance of herpes simplex virus type 1 thymidine kinase (TK(HSV1)) relative to the human isoenzyme. The enzyme selectively phosphorylates nucleoside analogs that can either inhibit viral DNA polymerase or cause toxic effects when incorporated into viral DNA. To relate structural properties of TK(HSV1) ligands to their chemical reactivity we have carried out ab initio quantum chemistry calculations within the density functional theory framework in combination with biochemical studies. Calculations have focused on a set of ligands carrying a representative set of the large spectrum of sugar-mimicking moieties and for which structural information of the TK(HSV1)-ligand complex is available. The k(cat) values of these ligands have been measured under the same experimental conditions using an UV spectrophotometric assay. The calculations point to the crucial role of electric dipole moment of ligands and its interaction with the negatively charged residue Glu(225). A striking correlation is found between the energetics associated with this interaction and the k(cat) values measured under homogeneous conditions. This finding uncovers a fundamental aspect of the mechanism governing substrate diversity and catalytic turnover and thus represents a significant step toward the rational design of novel and powerful prodrugs for antiviral and TK(HSV1)-linked suicide gene therapies.     

Purification and crystallization of thymidine kinase from herpes simplex virus type 1

Thymidine kinase from herpes simplex virus type 1 (ATP:thymidine 5'-phosphotransferase; EC 2.7.1.21) has been purified from an overexpression system and crystallized against ammonium sulfate by using the hanging-drop technique. The tetragonal crystals are of space group P4122 or P4322, and have unit cell dimensions a = b = 84 A, c = 180 A.     

Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.     

Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type II. II. Purification and characterization.

Deoxythymidine kinase activities were induced in HeLa TK- (deoxythymidine kinase-deficient) cells infected with either herpes simplex virus type I or herpes simplex virus type II. The herpes simplex virus type I-induced enzyme was found in the cytoplasmic and nuclear fractions of the infected cells, whereas the herpes simplex type II-induced deoxythymidine kinase could only be found in the cytoplasm. Herpes simplex virus type I and II specific deoxythymidine kinases were purified by affinity column chromatography. Both purified deoxythymidine kinases retained the deoxycytidine kinase activity present in the crude preparation. The purified herpes simplex virus type I deoxythymidine kinase had a different mobility on electrophoresis, but the same sedimentation rate on a glycerol gradient as the corresponding unpurified enzyme, whereas the purified herpes simplex virus type II deoxythymidine kinase had the same mobility and sedimentation rate as the corresponding unpurified enzyme. In the presence of Mg2+ATP and dithiothreitol, herpes simplex virus type II deoxythymidine kinase was more stable than herpes simplex virus type I deoxythymidine kinase at both 45 degrees and 4 degrees. The deoxycytidine kinase activity present in the purified preparations was inactivated at the same rate as the deoxythymidine kinase activity. In the presence of the other substrate, deoxythymidine, herpes simplex virus type I deoxythymidine kinase was more stable than herpes simplex virus type II kinase. The purified herpes simplex virus type I and II deoxythymidine kinase had different activation energies when Mg2+ATP and deoxythymidine were used as substrates, but showed the same sensitivity toward ammonium sulfate inhibition.     

Development and analysis of a transformation-defective mutant of Harvey murine sarcoma tk virus and its gene product.

The Harvey murine sarcoma virus has been cloned and induces focus formation on NIH 3T3 cells. Recombinants of this virus have been constructed which include the thymidine kinase gene of herpes simplex virus type 1 in a downstream linkage with the p21 ras gene of Harvey murine sarcoma virus. Harvey murine sarcoma tk virus rescued from cells transfected with this construct is both thymidine kinase positive and focus inducing in in vitro transmission studies. The hypoxanthine-aminopterin-thymidine selectability of the thymidine kinase gene carried by this virus has been exploited to develop three mutants defective in the p21 ras sequence. All three are focus negative and thymidine kinase positive when transmitted to suitable cells. Of these, only one encodes a p22 that is immunologically related to p21. This mutant has been used to explore the relationship between the known characteristics of p21 and cellular transformation. Data presented herein indicate that the p21 of Harvey murine sarcoma virus consists of at least two domains, one which specifies the guanine nucleotide-binding activity of p21 and the other which is involved in p21-membrane association in transformed cells.     

Inhibition of herpes simplex virus type 2-induced biochemical transformation by interferon.

The effect of interferon on the biochemical transformation of thymidine kinase-deficient cells by UV-inactivated herpes simplex virus type 2 has been studied. Transformation was much less sensitive to the action of interferon than virus multiplication. However, the continuous presence of a high dose of interferon (2,000 U) inhibited transformation almost completely. Although we could not differentiate between the effect of interferon on fixation and expression of the virus thymidine kinase gene, data suggest that the inhibitory effect of interferon on transformation might be partially due to the suppression of virus thymidine kinase expression.     

Novel rearrangements of herpes simplex virus DNA sequences resulting from duplication of a sequence within the unique region of the L component.

We constructed insertion mutants of herpes simplex virus type 1 that contained a duplication of DNA sequences from the BamHI-L fragment (map units 0.706 to 0.744), which is located in the unique region of the L component (UL) of the herpes simplex virus type 1 genome. The second copy of the BamHI-L sequence was inserted in inverted orientation into the viral thymidine kinase gene (map units 0.30 to 0.32), also located within UL. A significant fraction of the progeny produced by these insertion mutants had genomes with rearranged DNA sequences, presumably resulting from intramolecular or intermolecular recombination between the BamHI-L sequences at the two different genomic locations. The rearranged genomes either had an inversion of the DNA sequence flanked by the duplication or were recombinant molecules in which different regions of the genome had been duplicated and deleted. Genomic rearrangements similar to those described here have been reported previously but only for herpes simplex virus insertion mutants containing an extra copy of the repetitive a sequence. Such rearrangements have not been reported for insertion mutants that contain duplications of herpes simplex virus DNA sequences from largely unique regions of the genome. The implications of these results are discussed.     

Cloning, expression, isolation and properties of thymidine kinase herpes simplex virus, strain L2

A thymidine kinase UL23 gene (EC 2.7.1.145) from an acyclovir-sensitive strain L2 of herpes simplex virus type 1 was cloned and expressed in E. coli. Enzyme was purified by chromatography to a homogeneous state controlled by PAG electrophoresis. The Michaelis constants for the reactions with thymidine and an acyclovir were determined. It was found that enzyme phosphorilate some modified nucleosides such as d2T, d4T, d2C, 3TC, FLT, BVDU, GCV. A comparison of the purified enzyme properties and properties ofthymidine kinase of other strains of herpes simplex virus, previously published was carried out. It is shown that enzyme is inhibited by acyclovir H-phosphonate.     

A generalized technique for deletion of specific genes in large genomes: a gene 22 of herpes simplex virus 1 is not essential for growth

We describe a general method for inactivation and deletion of genes at specific sites in large DNA genomes. In the first step of the procedure, the herpes simplex virus thymidine kinase is inserted into the genome at a specific site. In the second step, the thymidine kinase gene and desired sequences flanking the insertion site are deleted. Both steps involve recombination of the genomes with cloned chimeric fragments and utilize the available selection for or against thymidine kinase to select the desired genomes. We have applied the procedure to inactivate and to delete portions of an a gene of herpes simplex virus 1 specifying protein 22. The recombinant virus carrying the thymidine kinase inserted into the gene 22 and viruses exhibiting 0.1 kb and 0.7 kb deletions in the gene 22 specify new α polypeptides with molecular weights approximately 30% of the wild-type gene 22 product and grown normally in Vero cell cultures.