David Trkula

Nucleotide sequence of the herpes simplex virus type 2 (HSV-2) thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide and its comparison with the HSV-1 thymidine kinase gene

To analyze the boundaries of the functional coding region of the HSV-2(333) thymidine kinase gene (TK gene), deletion mutants of hybrid plasmid pMAR401 H2G, which contains the 17.5 kbp BglII-G fragment of HSV-2 DNA, were prepared and tested for capacity to transform LM(TK-) cells to the thymidine kinase-positive phenotype. These studies showed that hybrid plasmids containing 2.2-2.4 kbp subfragments of HSV-2 BglII-G DNA transformed LM(TK-) cells to the thymidine kinase-positive phenotype and suggested that the region critical for transformation might be less than 2 kbp. That the activity expressed in the transformants was HSV-2 thymidine kinase was shown by experiments with type-specific enzyme-inhibiting rabbit antisera and by disc-polyacrylamide gel electrophoresis analyses. DNA fragments of the HSV-2 TK gene were subcloned in phage M13mp9 and M13mp8. A sequence of 1656 bp containing the entire coding region of the TK gene and the flanking sequences was determined by the dideoxynucleotide chain termination method. Comparisons with the HSV-1(Cl 101) TK gene revealed that PstI, PvuII, and EcoRI cleavage sites had homologous locations as did promoter, translational start and stop, and polyadenylation signals. Extensive homology was observed in the nucleotide sequence preceding the ATG translational start signal and in portions of the coding region of the genes. Comparisons of the predicted amino acid sequences of the HSV-1 and HSV-2 thymidine kinase polypeptides revealed that both were enriched in alanine, arginine, glycine, leucine, and proline residues and that clear, but interrupted homology existed within several regions of the polypeptide chains. Stretches of 15-30 amino acid residues were identical in conserved regions. The possibility is suggested that domains containing some of the conserved amino acid sequences might have a role in substrate binding and as major antigenic determinants.

Properties of mitochondrial thymidine kinases of parental and enzyme-deficient HeLa cells

Abstract HeLa(BU25), a mutant subline of HeLa S3 cells, contains mitochondrial thymidine (dT) kinase, despite a marked deficiency in the dT kinase activity of the “cytosol” (high-speed supernatant) cell fraction. The HeLa(BU25) mitochondrial dT kinase differs from the “cytosol” enzyme of parental HeLa S3 cells in sedimentation coefficient, ability to utilize ribonucleoside 5′-triphosphates other than ATP as phosphate donors, sensitivity to inhibition by dCTP, and in disc polyacrylamide gel electrophoretic (disc PAGE) patterns. Two dT kinase activities [relative mobilities (Rm) of 0.4 and 0.6–0.7] were detected after disc PAGE of HeLa(BU25) mitochondrial extracts and both activities migrated more rapidly than the typical cytosol enzyme (Rm = 0.2) of dT kinase-positive human cells. The 0.6 to 0.7-Rm dT kinase of HeLa(BU25) mitochondria, but not the 0.4-Rm activity, utilized GTP and UTP, as well as ATP, as phosphate donors. HeLa S3 mitochondrial fractions contained the 0.6–0.7 Rm and the 0.4-Rm activities, and in addition, a “cytosol-like” 0.2-Rm activity. The 0.6 to 0.7-Rm dT kinase of HeLa S3 mitochondria utilized either UTP or ATP as phosphate donors, but the 0.4- and 0.2-Rm dT kinases utilized only ATP. Similarly, the HeLa S3 cytosol dT kinase efficiently utilized ATP, but not UTP, as a phosphate donor.

Thymidine Kinases Induced by Avian and Human Herpesviruses

Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation experiments were carried out to characterize thymidine (dT) kinase isozymes induced by herpesvirus of turkeys and infectious laryngotracheitis virus in the cytosol fraction of infected chick cells. The avian herpesvirus dT kinases differed from chick cytosol dT kinase in electrophoretic mobility, isoelectric point and phosphate donor specificity. The avian herpesvirus dT kinases resembled chick mitochondrial dT kinase in electrophoretic mobilityy and isoelectric point, but exhibited larger sedimentation coefficients. The avian herpesvirus enzymes closely resembled dT kinases induced by human herpes simplex viruses types 1 and 2.

Sequential genital infections by herpes simplex viruses types 1 and 2: Restriction nuclease analyses of viruses from recurrent infections

Virus isolated from a woman presenting with the first symptomatic episode of genital herpes was identified as herpes simplex virus type 1 (HSV-1) by restriction nuclease fingerprinting. Testing for IgM antibody to HSV indicated that the patient had recently contracted a new HSV infection. Virus microneutralization and the micro-solid phase radioimmunometric test for IgG, however, showed that the patient had had prior infection with herpes simplex virus type 2 (HSV-2); thus the HSV-1 infection was acquired despite the presence of antibody to HSV-2. Genital herpes recurred about four, seven, and nine months after the HSV-1 infection. Isolates from the latter three episodes all were of an identical strain of HSV-2 and were not recombinants or a mixture of the viruses. The data show that two distinctly different herpes simplex viruses can initiate genital infections in one individual and suggest that HSV-2 is more likely to recur than HSV-1.

Mitochondrial thymidine kinase of bromodeoxyuridine-resistant, kinase-deficient HeLa(BU25) cells

Abstract Total cell extracts of HeLa(BU25), a mutant subline of HeLa S3, are deficient in dT kinase activity. In contrast to the parental HeLa S3 cells, the nuclear DNA of HeLa(BU25) does not exhibit a “heavy” density after the HeLa(BU25) cells are grown in medium with dBU. Despite the loss of the principal dU-dT phosphorylating enzyme, HeLa(BU25) cells contain a mitochondrial dU-dT phosphorylating activity with a specific activity about equal to that of the HeLa S3 mitochondrial enzyme. Phosphorylation of 3 H-dU by the mitochondrial enzyme requires ATP, is markedly inhibited by dTTP or nonradioactive dT, but not by Urd, Cyd, or Ado. Hence, it is a dT kinase. A nucleoside phosphotransferase, weakly active at pH 5.5–6.5, may also be present.

Distinctive properties of mitochondrial thymidine(dT)kinase from bromodeoxyuridine(dBU)-resistant mouse lines

Abstract dBU-resistant mouse lines lack detectable dT kinase activity in the high speed supernatant (cytosol) cell fraction. However, they contain a mitochondrial dT kinase, which sediments more slowly in glycerol gradients than the cytosol enzyme of parental mouse lines, exhibits a disc PAGE mobility relative to the tracking dye (Rm) of about 0.7–0.8, and utilizes ATP, UTP, GTP, and CTP as phosphate donors. The mitochondrial fraction of parental cells also contains this 0.7–0.8 Rm activity and, in addition, a minor dT kinase activity which migrates faster than the cytosol enzyme, but utilizes only ATP as phosphate donor. The cytosol dT kinase of parental mouse lines exhibits an Rm of about 0.2–0.3 and utilizes only ATP as phosphate donor.

Characterization of nucleoside phosphotransferase and thymidine kinase activities of chick embryo cells and of chick-mouse somatic cell hybrids

Abstract Experiments were carried out to characterize the thymidine (dT) phosphorylating activities of chick embryo, chick erythrocytes, and of chick mouse somatic cell hybrids derived from fused chick erythrocytes and dT kinase-deficient LM(TK) mouse cells. Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation analyses revealed that chick embryo cells contained four distinctive dT phosphorylating activities, two dT kinases and two nucleoside phosphotransferases. Thymidine kinase F. found principally in the cytosol, was also detected in mitochondrial and nuclear extracts, but was very low or absent from chick erythrocytes. Thymidine kinase A corresponds to the mitochondrial-specific isozyme found in bromodeoxyuridine-resistant mammalian cells. Nucleoside phosphotransferase activities were very active in chick embryo cytosol and were detected in embryo mitochondria! and nuclear extracts and cytosol and nuclear extracts of chick erythrocytes. Most of the chick embryo nucleoside phosphotransferase activity could be removed by purification of cytosol dT kinase F. Chick-mouse somatic cell hybrids exhibited chick dT kinase F, but neither chick dT kinase A. chick nucleoside phosphotransferase, nor mouse cytosol dT kinase activities. The results indicate (1) the genetic determinant for chick cytosol dT kinase F is on a different chromosome from the determinants for the chick nucleoside phosphotransferases and mitochondrial dT kinase A, and/or (2) only the chick cytosol dT kinase F, but neither the chick nucleoside phosphotransferases nor dT kinase A, was reactivated in the hybrids.

Subcellular Localization and Properties of Thymidine Kinase from Adenovirus-infected Cells

Summary The principal cytosol thymidine kinase activity of African green monkey kidney cells increased approximately threefold at 21 to 29 h after infection with adenovirus type 5. Disc polyacrylamide gel electrophoresis (disc PAGE) analyses showed that the electrophoretic mobility relative to the tracking dye (Rm) of the cytosol thymidine kinase from both mock-infected and adenovirus type 5-infected cells was about 0.23. The cytosol thymidine kinase from normal cells also resembled the cytosol enzyme from infected cells with respect to phosphate donor specificity and sedimentation coefficient. Mitochondria from normal and virus-infected cells contained a cytosol-like enzyme and, in addition, a distinctive mitochondrial isozyme exhibiting an Rm of about 0.6 and a smaller sedimentation coefficient than the cytosol enzyme. The activity of the mitochondrial-specific isozyme of thymidine kinase (Rm = 0.6) was not significantly increased by virus infection. The ratio of the two thymidine kinase activities found in mitochondria also was not markedly changed by virus infection. The results suggest that adenovirus type 5 infection reactivates an inactive molecular form of cytosol thymidine kinase or derepresses the synthesis of the cytosol enzyme, but not that of the mitochondrial-specific thymidine kinase. Adenovirus infection does not alter the electrophoretic mobilities of the cytosol and mitochondrial thymidine kinases.

Biochemical transformation of LM(TK−) cells by hybrid plasmids containing the coding region of the herpes simplex virus type 1 thymidine kinase gene

Abstract Recombinant plasmid pAGO codes for herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) and consists of a 2-kbp HSV-1 DNA fragment inserted at the unique Pvu II cleavage site of plasmid pBR322. A hybrid plasmid, designated pMH110, has been derived from plasmid pAGO by deleting the 1689-bp pBR322 nucleotide sequence of pAGO, which extends from the Bam HI to the Pvu II cleavage site, and the 250-bp HSV-1 nucleotide sequence of pAGO, which extends from the Pvu II to the Bgl II cleavage site. Plasmid pMH110 biochemically transformed LM(TK − )cells to the TK + phenotype. The biochemically transformed cell lines had the following properties: (i) they were resistant to the growth-inhibiting effects of 1 m M thymidine; and (ii) they expressed an HSV-1-specific TK activity. This HSV-1 TK activity was purified after labeling biochemically transformed cell lines [LM(TK − )/TF pMH110 E2 and LM(TK − )/TF pMH110 Hc2] with [ 35 S]methionine. Immunoprecipitation experiments revealed that the TK polypeptides made in the biochemically transformed cells had molecular weights of about 39,000 to 40,000, which are about the same as the molecular weights of the TK polypeptides previously purified from HSV-1-infected LM(TK − ) cells and other biochemically transformed cell lines. The experiments support the hypothesis that the functional coding region of the HSV-1 TK gene is 3′ to the Bgl II cleavage site, and they also suggest that the HSV-1 TK messenger RNA may have been initiated in cells transformed by Hinc II- and Eco RI-cleaved pMH110 DNA at a site in cellular (or plasmid) DNA upstream from the HSV-1 DNA Bgl II cleavage site.

Biochemical transformation of mouse cells by a purified fragment of marmoset herpesvirus DNA.

Although the size of marmoset herpesvirus (MarHV) DNA, estimated by velocity sedimentation in sucrose gradients, was similar to that of herpes simplex virus type 1 (HSV-1) DNA, the restriction endonuclease sites of MarHV and HSV-1 DNAs were quite different. A specific BamHI restriction fragment (6.2 x 10(6) daltons) of MarHV DNA biochemically transformed LM(TK-) mouse fibroblasts to the thymidine kinase(TK)-positive phenotype. Rabbit antisera, prepared against MarHV TK, inhibited MarHV-induced TK, but not HSV-1, HSV-2, or cellular TKs. Disc PAGE analyses and enzyme neutralization experiments with the anti-MarHV TK sera demonstrated that the TK expressed in MarHV transformants was MarHV-specific.