Deoxypyrimidine Kinase Research Articles

Unique metabolism of a novel antiviral L-nucleoside analog, 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil: a substrate for both thymidine kinase and deoxycytidine kinase.

2'-Fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU) is the first L-nucleoside analog with low cytotoxicity discovered to have potent antiviral activities against both hepatitis B virus and Epstein-Barr virus but not human immunodeficiency virus. This spectrum of activity is different from those of the other L-nucleoside analogs examined. L-FMAU enters cells through equilibrative-sensitive and -insensitive nucleoside transport as well as through nonfacilitated passive diffusion. L-FMAU is phosphorylated stepwise in cells to its mono-, di-, and triphosphate forms. In the present study the enzymes responsible for the first step of L-FMAU phosphorylation were identified. This is the first thymidine analog shown to be a substrate not only for cytosolic thymidine kinase and mitochondrial deoxypyrimidine kinase but also for deoxycytidine kinase. This finding suggests that the antiviral activity of L-FMAU will not be limited by the loss or alteration of any of these deoxynucleoside kinases.

The role of cytoplasmic deoxycytidine kinase in the mitochondrial effects of the anti-human immunodeficiency virus compound, 2‘,3‘-dideoxycytidine.

2',3'-Dideoxycytidine (ddC) is a potent inhibitor of human immunodeficiency virus replication in vitro and shows beneficial effects in AIDS therapy. The compound inhibits mitochondrial DNA (mtDNA) synthesis at a clinically relevant concentration, which could be responsible for the side effects of ddC observed in the clinic. Thymidine (dThd), one of the substrates of mitochondrial deoxypyrimidine kinase (dPyd kinase), was not able to reverse the mitochondrial toxicity of ddC in CEM cells. Furthermore, the cytoplasmic deoxycytidine kinase (dCyd kinase)-deficient CEM cells were highly resistant to the mitochondrial toxicity of ddC. These data suggest a critical role for cytoplasmic dCyd kinase in the mitochondrial toxicity of ddC. The metabolites of ddC, but not ddC itself, were able to inhibit mtDNA synthesis in isolated mitochondria. The potency of the inhibitory effect was in the order of ddCTP greater than ddCDP greater than ddCMP greater than ddC. The lack of inhibition by ddC of mtDNA synthesis could be due to the inefficient ddC phosphorylation in mitochondria. Although the mitochondrial dPyd kinase was reported to phosphorylate ddC, the phosphorylation of ddC in isolated mitochondria was not detectable. The data suggest that ddC is phosphorylated to ddCTP in the cytoplasm and then transported into mitochondria to exert its inhibitory effect on mtDNA synthesis.

Quaternary structure of vaccinia virus thymidine kinase

Thymidine kinase enzymes isolated from a variety of sources are generally considered to have a native molecular weight of 80–90 kDa composed of two 40–45 kDa subunits. Although these parameters may accurately describe the atypical deoxypyrimidine kinases expressed by members of the Herpesviridae, the nucleotide sequences of thymidine kinase genes isolated from human, mouse, chicken and a variety of poxviruses (vaccinia virus, monkeypox virus, variola virus, fowlpox virus and capripoxvirus) predict molecular weights on the order of 20–25 kDa for the derived primary translation products. To resolve this apparent dilemma, velocity sedimentation centrifugation, gel filtration chromatography and protein cross-linking procedures were employed to provide experimental evidence that enzymatically-active vaccinia virus thymidine kinase is a homotetrameric complex of 20 kDa monomers with a native M r of 80 kDa.

Vaccinia virus encodes a thymidylate kinase gene: sequence and transcriptional mapping

The nucleotide sequence and deduced amino acid sequence of a vaccinia virus gene from the SalI F fragment are shown. The predicted polypeptide shares 42% amino acid identity over a 200 amino acid region with Saccharomyces cerevisiae thymidylate kinase (TmpK) and has low homology with herpes simplex virus deoxypyrimidine kinase. Northern blotting and S1 nuclease protection showed that the TmpK gene is transcribed early during infection and mapped the mRNA 5' end to immediately upstream of the second inframe ATG codon of the open reading frame (ORF). The encoded polypeptide is predicted to be 204 amino acids long (23.2 kD) and is almost colinear with yeast TmpK. Vaccinia virus possesses genes for TK and TmpK, separated by 57 kilobases of DNA, which are co-ordinately expressed and the encoded enzymes perform sequential steps in the same biochemical pathway.

Distinctive multiple thymidine kinase species of the aquatic filamentous fungus Achlya ambisexualis

Four thymidine kinase (TK) species were found in the cell extract of the filamentous fungus Achyla ambisexualis. They differed by their relative electrophoretic mobilities (Rm) on polyacrylamide gel in nondenaturing buffer and are designated as the Rm 0.30, 0.45, 0.65, and 0.85 TK species. The Rm 0.30 activity was readily solubilized from the mycelia by freeze–thaw treatment, whereas the other TK species were solubilized by additional treatment with nonionic detergent nonidet P-40. The Rm 0.30, 0.65, and 0.85 TK species utilized only ATP as a phosphate donor and dT as a phosphate acceptor. However, the Rm 0.45 species utilized all four ribonucleoside triphosphates as phosphate donors and dC, in addition to dT, as phosphate acceptors. Therefore, this TK species is a deoxypyrimidine kinase. All four TK activities were inhibited by the feedback inhibitor dTTP. However, only the Rm 0.45 TK activity was inhibited by dCTP. The characterization of distinctive multiple TK species in A. ambisexualis will facilitate the studies using this fungus as a transformation host cell for vectors carrying TK as a reported gene.

CHARACTERIZATION OF ANTIBODIES TO VARICELLAZOSTER VIRUS BY WESTERN BLOT

The immune response to varicella-zoster virus (VZV) includes antibody (Ab) to virally-specified proteins (P) and glycoproteins (GP). We analyzed Ab after chickenpox (CP), zoster (Z), and live attenuated varicella vaccine (LAW) by western blot (WB). Responses to 2 of the 3 major GPs were identified by comparing bands on standard WBs using infected VZV cell lysate as antigen with a reference WB in which Z serum was reacted with purified GP I and GP II. A mouse monoclonal Ab to GP I confirmed its location, and all 3 GPs were visualized on silver stain of SDS-polyacrylamide gel. Preimmune sera and an uninfected cell lysate were controls. All convalescent sera from patients with CP(N=5) had antibody to GP I (bands at 99 and 92 kilodaltons [kd]), GP II (125 kd), and GP III (113 kd). In addition 5/5 had a strong band at 147 kd and 4/5 had a band at 39 kd, near the molec. wt. of VZV deoxypyrimidine kinase. Bands were well-preserved to GP I in 5 remote CP sera, but faint at GP II and III and at 147 kd. In 7/8 pts. post Z, bands at all 3 GPs and at 39 kd were darker than after recent CP despite equivalent fluorescent antibody to membrane antigen (FAMA) liters. Z sera also had bands at 51, 48, 46, and 43 which were absent or faint post CP. After 2 doses of LAW, 10/11 vaccinees had bands at GP I. Fewer (6/11) had a band at GP II and only 3/11 had a band at GP III. However.the WBs of 4 vaccinees after breakthrough CP were similar in intensity and band location to WBs post Z. Ten leukemic children who lost FAMA Ab after LAW and later reseroconverted without clinical illness also had WBs resembling the anamnestic profile seen post Z. Even when FAMA negative, these silent FAMA reconverted were protected from serious CP. WB is useful in distinguishing secondary from primary immune response to VZV.

Characterization of an Epstein-Barr virus-induced thymidine kinase.

Previous work from our laboratory suggested that the selective inhibition of Epstein-Barr virus (EBV) replication by 1-beta-D-arabinofuranosylthymine in human lymphoid cell lines involved the induction of a new thymidine kinase (TK) able to phosphorylate the thymidine analog. We further characterized this enzyme induced in various EBV-positive cell lines after viral genome activation with a combination of sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate. The following results confirmed the existence of an EBV-specific deoxypyrimidine kinase: induction of EBV-related TK was connected with the appearance of viral early antigens in EBV-carrying cells; unexpected behaviors of the enzyme activity upon different fractionating treatments led to the conclusion that EBV-induced TK was extracted as a complex molecular form, larger than other known cellular or viral isozymes; enzymatic properties distinguished EBV-induced TK from host lymphoid cell isozymes but made it resemble other herpesvirus-specific deoxypyrimidine kinases, i.e., by partial inhibition by dTTP or ammonium sulfate, insensitiveness to dCTP, and nonstringent specificity for normal TK substrates. Genetic evidence is required to definitively ensure that EBV-specific TK actually is virus coded in EBV-transformed human lymphoid cells.

Mapping of the varicella zoster virus deoxypyrimidine kinase gene and preliminary identification of its transcript

The varicella-zoster virus (VZV) deoxypyrimidine kinase (dPK) gene was mapped by transfection of cloned viral DNA fragments into thymidine kinase-deficient mouse L (LTK −) cells and subsequent biochemical transformation of these cells to the LTK + phenotype. Such transforming activity was limited to the BamHI-H and EcoRI-D fragments of the VZV genome, which overlap by 2.2 kb between map units 0.50 and 0.52. Biochemically transformed cells were shown to contain a high copy number of viral DNA sequences that had integrated into the cellular DNA. Extracts of these cells showed a higher level of dPK activity than did extracts of parental LTK − cells. With the use of Northern hybridization analysis of transformed and VZV-infected cell RNAs, it was possible to tentatively assign a 1.8-kb transcript to the VZV dPK.

Carrier mediated uptake of deoxyguanosine in rat liver mitochondria.

It has been known for a number of years that mitochondria contain enzymes important for the synthesis of DNA. A distinct mitochondrial deoxyguanosine kinase was first documented by Gower etal. (1) in calf thymus. Mitochondrial deoxycytidine/deoxythymidine kinase (2) was demonstrated in blast cells of leukemia patients and HeLa mitochondria contain a deoxythymidine kinase (3). Mouse, human and chick mitochondria possess a deoxypyrimidine kinase (3). A recent report by Fabianowska-Majewska and Greger (4) shows that rat liver mitochondria contain deoxythymidine, deoxcytidine, deoxyadenosine and deoxyguanosine kinase activities, however few details regarding these activities were reported.

Inhibition of the herpes simplex virus-coded thymidine kinase-complex by 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) and 9-(2-hydroxyethoxymethyl)guanine-monophosphate (acyclo-GMP).

The thymidine kinase-complex isolated from herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) is associated with the following enzyme activities:ATP:dThd (dCyd) deoxypyrimidine kinase, ATP:dTMP thymidylate kinase, ADP:dThd- and AMP:dThd 5'-phosphotransferase. In kinetic experiments it is shown that ara-AMP inhibits AMP:dThd- and ADP:dThd phosphotransferase activity, while acyclo-GMP impairs ADP:dThd phosphotransferase reaction only; the inhibition was found to be non-competitive. The functional subunit ATP:dThd kinase was not affected by either compound.

Synthesis of some 5′-amino-2′,5′-dideoxy-5-iodouridine derivatives and their antiviral properties against herpes simples virus

In an attempt to improve the antiviral efficacy of 5′-amino-2′,5′-dideoxy-5-iodouridine (AIdU) the N-acetyl and N,3′- O-diacetyl derivatives were prepared. N-Acetylation of AIdU increased its ability to inhibit the phosphorylation of thymidine by the deoxypyrimidine kinase of herpes simplex virus type 1 (HSV1) while diacetylation had the converse effect. The affinity of the corresponding compounds containing uracil or thymine for virus deoxypyrimidine kinase was also determined. A range of N-acyl-, N-sulphonyl- and N,3′- O-diacyl- derivatives of AIdU were synthesised; enhanced inhibition of deoxypyrimidine kinase by a number of these compounds was observed. The previous observation that 5′-azido-2′,5′-dideoxy-5-iodouridine has antiherpetic activity in vivo led us to investigate its 3′- O-acetyl derivative as well as the corresponding compound containing uracil. None of the derivatives described showed antiviral activity in cell culture against HSV1; acylation failed to enhance the potency of AIdU against HSV1 in vivo.

Adenosine diphosphate: Thymidine 5′-phosphotransferase, a new enzyme activity, associated with the herpes simplex virus-induced deoxypyrimidine kinase

The deoxypyrimidine kinase induced in mouse fibroblasts, strain CLID (TK −) infected with either herpes simplex virus (HSV) type 1 or type 2, possesses besides deoxypyrimidine kinase (ATP:dThd/dCyd phosphotransferase) two further enzyme activities: an AMP:dThd phosphotransferase and an ADP:dThd phosphotransferase. The latter enzyme activity, described in this report, was found to be inhibited by antiserum against the HSV deoxypyrimidine kinase and to be absent after infection with TK − mutant MDK 10 (B 2006). The ADP:dThd phosphotransferase, which had been purified approx. 340-fold, differs by a series of physicocbemical properties from the viral AMP:dThd- and ATP:dThd phosphotransferase.

Chemotherapy of herpetic keratitis induced by acyclovir-resistant strains of herpes simplex virus type 1

The severity of herpetic keratitis induced by 9-(2-hydroxyethoxymethyl) guanine-resistant strains of herpes simplex virus was significantly reduced by cotherapy with 9-β-D-arabinofuranosyladenine (ara-A) and 2-deoxycoformycin. Therapy with 5-trifluoromethyl-2′-deoxyuridine (F 3TdR) significantly reduced the severity of keratitis induced by an acyclovir-resistant strain with a defective DNA polymerase. Therapy with 3 percent acyclovir ointment slightly reduced the number of herpetic lesions produced by either deoxypyrimidine kinase or DNA polymerase defective viruses, despite these viruses being 100 to 1000 times more resistant to acyclovir than the wildtype strain. Therapy with 3 percent ara-A ointment alone significantly reduced the severity of lesions produced by the wildtype herpes strain. Therapy with ara-A alone did not reduce the severity of disease induced by any of the acyclovir-resistant mutants. The sensitivity of the wildtype and mutant viruses to nucleoside analogs was confirmed by yield-reduction assays conducted with Vero cells. These studies indicate that cotherapy with ara-A and an adenosine deaminase inhibitor was a reasonable alternative therapy for keratitis due to mutants resistant to therapy with nucleoside analogs which require the virus-specified deoxypyrimidine kinase or DNA polymerase, while ara-A alone was not an effective alternative.

Adenylic Acid: Deoxythymidine 5'-Phosphotransferase: Evidence for the Existence of a Novel Herpes Simplex Virus-induced Enzyme

BHK (dPyK-) cells infected with herpes simplex virus type 1 (HSV-1) contain a virus-induced deoxythymidine (dThd)-phosphorylating enzyme. This enzyme uses AMP as phosphate donor and is called AMP :deoxythymidine 5'-phosphotransferase (or kinase). The enzyme was purified over 1300-fold and was found to be specific for an AMP substrate. It can thus be distinguished from virus-specific deoxypyrimidine kinase (dPyK). It is shown that the two substrates AMP and dThd participate in the reaction at a 1 : 1 molar ratio; the Km for AMP was 2 X 3 muM and for dThd it was 2 X 1 muM. The mol. wt. of the enzyme was estimated to be between 110 000 (by glycerol gradient centrifugation) and 90 000 (by gel filtration). For optimum activity, the phosphotransferase required an alkaline pH, and 37 degrees C; the activation energy of the reaction was 18 450 cal/mol. The appearance of the enzyme after infection parallels that of viral DNA synthesis-related functions.

Resistance of herpes simplex virus to acycloguanosine—genetic and physical analysis

Resistance of herpes simplex viruses (HSV-1 and HSV-2) to acycloguanosine (ACG) is determined by the two genetic loci of HSV coding for deoxypyrimidine kinase (thymidine kinase) and DNA polymerase activity. Mutants of HSV-1 which induce defective deoxypyrimidine kinase (dPyK − mutants) activity can participate in interallelic complementation, and show variability in their resistance to acycloguanosine (ACG R). This allows for subdivision of dPyK − mutants based on their resistance to ACG. Mutants dPyK − and dPyK −11 are in one interallelic complementation group and both are 100-fold more resistant to ACG in a plaque reduction assay than is wild type herpes simplex virus type 1 (HSV-1). (The 50% inhibitory concentrations (ID 50) are 6.0 and 7.0 μM of ACG compared to 0.06 μM.) Mutant MDK (Kit) which cannot complement any other mutant is 500-fold more resistant to ACG (ID 50 = 50 μM). Other dPyK − mutants fall in between in their sensitivity to ACG. The map position of the mutations in the DNA polymerase locus which result in resistance to ACG ( acg r) was located by correlating the ACG sensitivity of HSV-1IHSV-2 intertypic combinants with restriction endonuclease analysis of their DNA. The mutations acg r and paa r (phosphonoacetic acid resistance) are contained in the same 1.3 kbp region of DNA (map units 40.2 to 41.0). The level of sensitivity to ACG is type specific, differing in HSV-1 and HSV-2. This type specificity is determined by DNA sequences within map units 39.6 to 41.0. Thus the DNA sequences for type-specific sensitivity and resistance to ACG are shown to be distinct and clustered in the same region as the HSV DNA polymerase locus.

Cooperation between herpes simplex virus specific alpha protein and host cell RNA polymerase II in the transcription of viral deoxypyrimidine kinase.

A cistron specific enzyme-forming capacity method was used to study the control of herpes simplex virus (HSV) specific deoxypyrimidine kinase (dPyK) mRNA synthesis. In this assay, the alpha (or immediately early) protein was required to effect the transcription of dPyK mRNA. However, the dPyK mRNA synthesis was sensitive to alpha-amanitin in alpha-amanitin sensitive cells and resistant to alpha-amanitin in alpha-amanitin resistant cells. The effective dose range of alpha-amanitin used and the genetic lesion in alpha-amanitin resistant cells suggested that cellular DNA-dependent RNA polymerase II was also involved in the transcription of dPyK. This study suggests that two components, the HSV alpha protein and the cellular RNA polymerase, II, were required for dPyK mRNA synthesis.

The Effect of 1-beta-D-Arabinofuranosylthymine on the Growth of Herpes Simplex Virus Types 1 and 2

The influence of araT on the synthesis of HSV of types 1 and 2 including a TK(--) variant, on the activity of the dThd and the dCyd kinases and on the incorporation of radioactivity into insoluble or soluble material after infection was studied. AraT inhibited different parameters of the replication of a TK(--) variant; it is possibly phosphorylated by enzymes other than the deoxypyrimidine kinase and therefore it is less selectively phosphorylated than dThd. The mode of action of araT on herpes replication must be studied in more detail.

Cell-free Synthesis of Herpes Simplex Virus DNA: Studies with Three Temperature-sensitive Mutants of HSV Type 1

Herpes simplex virus (HSV) has a number of features which suggest that this virus might be ideally suited for a combined biochemical and genetic approach to DNA replication in a mammalian system. After infection of permissive cells, HSV rapidly reduces host-cell macromolecular synthesis and substitutes a large number of virus-coded enzymes related to DNA replication. Among them are a deoxypyrimidine kinase (Kit and Dubbs 1963; Jamieson et al. 1974) and other enzymes involved in precursor metabolism, a new DNA polymerase (Weissbach et al. 1973; Powell and Purifoy 1977), and an alkaline exonuclease (Keir and Gold 1963; Francke et al. 1978). It is rather likely that several other virus-coded functions, not yet identified biochemically, are required for the replication of viral DNA. This is borne out by the large number of DNA-negative complementation groups identified genetically (Subak-Sharpe et al. 1975; Benyesh-Melnick et al. 1975). The long range goal of the work...

Evidence for a herpes simplex virus-specific factor controlling the transcription of deoxypyrimidine kinase

A cistron-specific, enzyme-forming-capacity method was used to study the control of herpes simplex virus (HSV)-specific deoxypyrimidine kinase (dPyK) mRNA synthesis. A virus-specific factor was formed by a primary infecting virus, and this factor effected the transcription of dPyK mRNA of a superinfecting virus in the presence of cycloheximide, suggesting that the factor acted in "trans" and was a diffusible one. After the addition of actinomycin D to prevent further transcription and upon removal of cycloheximide, the dPyK mRNA was allowed to express into dPyK activity. A factor from HSV-1 could effect the transcription of dPyK mRNA of both HSV-1 and HSV-2. Amino acid analogs, canavanine or ethionine, inhibited the action of this factor, suggesting that a protein was involved. This protein factor was shown to belong to the alpha (or immediate-early) group of HSV-Specific polypeptides in preductively infected cells.

Transfer of genetic information via isolated amphibian metaphase chromosomes

The metaphase chromosome transfer system of McBride and Ozer (1973) has been adapted to a haploid, euploid, frog cell line. Genes coding for a deoxypyrimidine kinase and an enzyme responsible for a thymidine-specific saturable transport system have each been transferred at frequencies between 10(-6) and 10(-5) transferents per cell treated. Revertants for each of these two genes were observed at frequencies between 10(-8) and 10(-7) revertants per cell tested. Selfing controls showed no transferents. Two colonies were obtained in which cotransfer of both genes may have occurred. Activities of the transferred genes were assayed by incorporation of [3H]thymidine into alkali-stable, acid-precipitable material. Growth properties of 13 transferents in various media were also determined and presence of the appropriate enzymes inferred. These transferents were tested for stability early (25 generations) after transfer and were found to be stable. All 13 transferents possess the normal haploid number of chromosomes (n = 13) with no cytologically detectable chromosomal fragments.