Evaluation of thymidine kinase and neomycin phosphotransferase II positive selection systems for recovery of genetically atypical and recombinant DNA vaccine viruses

Dominant phenotypic marker cell culture selection systems were evaluated for the recovery of thymidine kinase positive (TK+), thymidine kinase negative (TK-), and neomycin phosphotransferase II positive (NPT II+) viruses. From 1 to 100 pfu of each marker-positive virus was diluted into 10(6) pfu of marker-negative background virus prior to selection. All three selection systems recovered 100 or fewer pfu of marked virus from the background population (10(-4) fractional level). In some instances, 1 to 10 pfu of marked virus were recovered (10(-5)-10(-6) fractional levels). TK+ and NPT II+ selections yielded nearly pure marker positive virus (98.8% and 99.5% respectively). Populations surviving TK- selection were significantly less pure (58.7%, P less than 0.05). Purity of recovered viruses was independent of the initial fractional level of marker-positive virus. The use of TK+, TK-, and NPT II+ selection systems is discussed in the context of purity testing and environmental surveillance of recombinant DNA viral vaccines.     

Genetic transformation of somatic cells. VII. The loss of the transformant phenotype is accompanied by both stable and unstable changes in DNA

From 6 clones of Chinese hamster cells varying in the rate of the loss of transformant phenotype and containing a thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1), 25 subclones negative in thymidine kinase (TK-) were isolated on a medium with 50 micrograms/ml 5-bromodeoxyuridine (BrdU). A study was made of the frequency of spontaneous reversions to the TK+ phenotype in cell populations of BrdU-resistant subclones, and of the transforming activity (upon transformation of TK- cells of A238 clone to the TK+ phenotype) of DNA preparations from a row BrdU-resistant subclones. In 7 of 11 BrdU-resistant subclones the TK- phenotype is associated with changes reducing significantly the transforming activity of DNA. Some of these alterations are stable and undergo no spontaneous reversion, while the other ones are unstable, being reversed or suppressed at a high frequency. BrdU-resistant subclones produced from clones more stable in transformant phenotype are on the whole more stable in the TK- phenotype than BrdU-resistant subclones from the clones with the high rate of the loss of the TK+ phenotype.     

Selective assay for herpes simplex viruses expressing thymidine kinase.

A technique for selecting herpes simplex viruses expressing the viral thymidine kinase (TK+) from a population of predominantly TK- viruses was developed. This was accomplished by infecting TK- cells and incubating the cultures under a liquid overlay medium containing methotrexate. Since the TK- cells survive in this medium for only a limited period of time, it was necessary to add fresh uninfected TK- cells 48 h after infection. The technique allowed the detection and quantitation of the TK+ virus fraction in mixtures of TK+ and TK- viruses where the TK+ fraction was present in frequencies as low as 10(-5). It was also used to estimate reversion frequencies and to obtain and analyze TK+ revertants from TK- mutant strains of herpes simplex virus type 1.     

Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.

We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli beta-galactosidase (beta gal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK- recombinants could be selected by a plaque assay on TK- cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous TK- mutants by the addition of a beta-gal indicator to the agarose overlay. Plaques that expressed beta-gal stained dark blue within several hours at 37 degrees C. Alternatively, TK- selection could be eliminated, and recombinant plaques could be readily identified solely by their blue color. The reverse procedure, in which the starting virus expresses beta-gal (i.e., forms blue plaques) and the desired recombinant has deleted the entire beta-gal gene (i.e., forms white plaques), is another alternative. Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.     

Complementary lethal invasion of the central nervous system by nonneuroinvasive herpes simplex virus types 1 and 2.

It is well-known that viral thymidine kinase (TK) expression is important for the maximum demonstration of virulence of herpes simplex virus (HSV). In this study, we have investigated interactions of a TK- mutant of virulent HSV type 2 (HSV-2) (syn+) and a nonneuroinvasive HSV-1 (syn) in mice. When the mice were inoculated with each virus alone in their rear footpads, no mice were killed even after infection with high doses of viruses (greater than 10(6) PFU per mouse), whereas 100% of the mice died when inoculated with 10(5) PFU of a 1:1 mixture of HSV-2 TK- mutant and nonneuroinvasive HSV-1. The 1:1 mixture exhibited even more virulence than the parental HSV-2; the mean survival time of coinfected mice was significantly shorter than that of mice inoculated with 10(5) PFU of the virulent HSV-2. We have also examined the genotypes and phenotypes of viruses isolated from the central nervous system of coinfected mice. Of 50 isolates, 7 were judged to be recombinants from their restriction endonuclease cleavage patterns, but all were nonneuroinvasive. In addition, all syn+ viruses (23 clones) tested were found to have TK- phenotypes, indicating that the majority of viruses present in the central nervous system were TK- viruses, since about 90% of viruses detected in spinal cords and brains exhibited syn+ phenotypes. These results strongly suggest that the lethal invasion of the central nervous system by HSV-2 TK- and nonneuroinvasive HSV-1 was the consequence of in vivo complementation between the two viruses.     

Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells.

The incorporation of [3H]thymidine into the deoxyribonucleic acid (DNA) of Chlamydia psittaci (strain 6BC) growing in thymidine kinase (adenosine 5'-triphosphate-thymidine 5'-phosphotransferase, EC 1.7.1.21)-containing L cells, L(TK+), and thymidine kinase-deficient L cells, LM(TK-), was examined by autoradiography. Label was detected over C. psittaci inclusions in L(TK+) but not LM(TK-) cells. No evidence for a chlamydia-specific thymidine kinase activity in either L(TK+) or LM(TK-) cells was obtained. Entry of [3H]thymidine into the DNA of C. psittaci growing in L(TK+) cells was quantitated by measuring label in purified C. psittaci. It was 265 times less efficient than entry into infected host cell DNA. It is concluded that low levels of exogenous thymidine are incorporated into the DNA of C. psittaci and that this incorporation is dependent on a fully competent host thymidine kinase activity. Evidence also is presented that L cells possess at least two thymidine kinase activities, both of which are capable of supplying thymidylate precursors for nuclear DNA synthesis.     

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.     

Thymidine kinase activity and trifluorothymidine resistance of spontaneous and mutagen-induced L5178Y cells in RPMI 1640 medium

L5178Y/TK+/- cells were treated with 3-methylcholanthrene (3MC) in order to obtain thymidine-kinase-deficient mutants (TK-/-) which were resistant to trifluorothymidine (TFTr). Clones of TK-/- cells were harvested from soft agar and adapted to growth in suspension culture. The phenotype of the TK-/- and TK+/- clones was confirmed by measuring thymidine kinase activity. These studies were undertaken with cells from 16 3MC-induced large colony clones (lambda TK-/-), 21 3MC-induced small colony clones (sigma TK-/-), and 51 spontaneous sigma TK-/- clones. Thymidine kinase activity was absent in all of the lambda TK-/- and sigma TK-/- 3MC-induced clones and also in 49 of 51 sigma TK-/- spontaneous clones. After at least 50 generations in suspension culture, TFTr was retained by 80% of the 3MC-induced lambda TK-/- cells, by 75% of the 3MC-induced sigma TK-/- cells, and by 89% of the spontaneous sigma TK-/- cells. The collective results showed that 86 of the 88 TFTr colonies examined lacked thymidine kinase activity and indicate that at least 98% of all TFTr colonies seen in the L5178Y assay are true TK-/- mutants.     

BK virus-plasmid expression vector that persists episomally in human cells and shuttles into Escherichia coli.

We describe a novel expression vector, pBK TK-1, that persists episomally in human cells that can be shuttled into bacteria. This vector includes sequences from BK virus (BKV), the thymidine kinase (TK) gene of herpes simplex virus type 1, and plasmid pML-1. TK+-transformed HeLa and 143 B cells contained predominantly full-length episomes. There were typically 20 to 40 (HeLa) and 75 to 120 143 B vector copies per cell, although some 143 B transformants contained hundreds. Low-molecular-weight DNA from TK+-transformed cells introduced into Escherichia coli were recovered as plasmids that were indistinguishable from the input vector. Removal of selective pressure had no apparent effect upon the episomal status of pBK TK-1 molecules in TK+-transformed cells. BKV T antigen may play a role in episomal replication of pBK TK-1 since this viral protein was expressed in TK+ transformants and since a plasmid that contained only the BKV origin of replication was highly amplified in BKV-transformed human cells that synthesize BKV T antigen.     

Genetic linkage but independent expression of functional HSV-1 tk and mammalian aprt genes after cotransfer to L cells

DNA-mediated gene transformation of mouse Ltk-aprt-hprt-cells was used to obtain stable, doubly selected transformants simultaneously expressing herpes virus thymidine kinase (TK) and mammalian adenine phosphoribosyltransferase (APRT). Cotransformants occurred at a frequency of 5 X 10(-6), a similar frequency for the transfer of the aprt marker has been previously observed. Isozyme and Southern blot analysis show that the TK and APRT expressed in these transformants resulted from gene transfer. For one stable cotransformant, [3H]thymidine [( 3H]TdR) selection against TK activity resulted in the loss of APRT activity as well, suggesting that these genes had become genetically linked together. Similarly selection against APRT expression resulted in the loss of a subset of the transferred herpes simplex virus tk genes. 5-Bromodeoxyuridine (BUdR) selected TK- variants differed from [3H]TdR selected TK- variants, in that they retained tk genes. However, BUdR-selected variants expressed full levels of APRT. Therefore, even though the transferred tk and aprt genes had become genetically linked together, they were, in this case, independently expressed since these cells were phenotypically TK- and APRT+.     

Phenotypic switching in cells transformed with the herpes simplex virus thymidine kinase gene

Biochemical transformation of Ltk- cells with the herpes simplex virus thymidine kinase (tk) gene resulted in numerous TK+ colonies that survived selection in hypoxanthine-aminopterin-thymidine medium. Many of these TK+ cell lines switched phenotypes and reverted to the TK- state. In this report, we describe the biological and biochemical characteristics of three TK- revertant lines. One (K1B5) transiently expressed TK in the presence of bromodeoxyuridine, which selects for the TK- phenotype. Another TK- sibling (K1B6n) expressed TK only after removal from bromodeoxyuridine-containing medium. The last variant (K1B6me) lost the ability to switch to the TK+ phenotype, although it maintained the herpes simplex virus sequences coding for TK. Loss of the ability of K1B6me cells to express TK was correlated with extensive methylation of the sequence recognized by the restriction endonuclease HpaII (pCpCpGpG). After these cells were treated with 5-azacytidine, they regained the ability to clone in hypoxanthine-aminopterin-thymidine medium and reexpressed virus tk mRNA and enzyme. In addition, the HpaII sites that were previously shown to be refractile to enzyme digestion were converted to a sensitive state, demonstrating that they were no longer methylated.     

Synthesis and evaluation of thymine-derived carboxamides against mitochondrial thymidine kinase (TK-2) and related enzymes

Based on the structure of our previously identified mitochondrial thymidine kinase (TK-2) inhibitors, three series of thymine-derived carboxamides have been synthesized and tested against TK-2 and related enzymes. The methodology employed has been a solution-phase parallel synthesis based on the coupling of three thymine-derived acids [4-(thymin-1-yl)butyric acid (I), [4-(thymin-1-yl)-butyrylamino]acetic acid (II) and 6-(thymin-1-yl)hexanoic acid (III)] with different commercially available primary amines that carry cyano and/or phenyl groups. The couplings were performed in good yields (from 60% to 90%), with the exception of those that incorporate the highly crowded triphenylmethylamine (e). From the new synthesized compounds, the N-trityl-6-(thymin-1-yl)hexanamide (IIIe) was the most active TK-2 inhibitor (IC(50)=19+/-2microM).     

Genetic transformation of somatic cells. VIII. The effect of the DNA methylation inhibitor 5-azacytidine on the stability of thymidine kinase-negative and -positive phenotypes of transformant clone cells

A study was made of the effect of an DNA methylation inhibitor 5-azacytidine (azaC) on the frequency of reversion to a thymidine kinase-positive (TK+) phenotype in 5-bromodeoxy-uridine (BrdU)-resistant subclones obtained from clones of Chinese hamster cells transformed by thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1). It is shown that in 8 of 15 BrdU-resistant subclones azaC increases 2-1000-fold the frequency of reversion to TK+ phenotype. Variations in the inducibility of reversions to TK+ phenotype indicate that the DNA methylation associated with TK- phenotype affects but differently tk gene of HSV1. Cultivation of TK+ cells of transformant clones in the presence of azaC may lead to stabilization (or decrease in the rate of the loss) of TK+ phenotype, or may not influence the stability of transformant phenotype. The reaction of TK+ cells of transformant clones depends both on genetically determined rate of the loss of TK+ phenotype, and on the structure of transforming DNA introduced to cells. A conclusion is drawn that the TK- phenotype of transformant clone cells arises due to processes which are not associated with methylation of tk gene of HSV1 in spite of the fact that such a methylation may later stabilize significantly the TK- phenotype.     

Murine mammary FM3A carcinoma cells transformed with the herpes simplex virus type 1 thymidine kinase gene are highly sensitive to the growth-inhibitory properties of (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds

Murine mammary carcinoma (FM3A TK-/HSV-1 TK+) cells, which are thymidine kinase (TK)-deficient but have been transformed with the herpes simplex virus type 1 (HSV-1) TK gene are inhibited in their growth by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) at 0.5, 0.5 and 0.8 ng/ml, respectively; i.e., a concentration 5000 to 20 000-fold lower than that required to inhibit the growth of the corresponding wild-type FM3A/0 cells. Hence, transformation of tumor cells with the HSV-1 TK gene makes them particularly sensitive to the cytostatic action of BVDU and related compounds.     

Genetic transformation of somatic cells. II. An analysis of the status of the plasmid nucleotide sequences in chromosomal DNA and the thymidine kinase activity in transformant clone cells

Chinese hamster A238 TK- -cells were transformed with plasmids (derivatives of pBR325) containing thymidine kinase (TK) gene of Herpes simplex virus type 1 (HSV1). The results of dot- and blot-hybridization indicate the presence of pBR325 sequences in the chromosomal fractions of DNA in the transformant clones. These sequences are probably tandemly arranged, and each cluster contains 25--50 copies. SV40 sequences cloned in pBR325 were introduced into the Chinese hamster cells by co-transformation with TK-gene of HSV1-containing plasmid DNA, and all the co-transformant clones selected for TK+-phenotype were shown by hydridization to contain 3V40 DNA fragments. Isoelectrofocusing in polyacrylamide gel shows that thymidine kinase from TK+-transformant clones is of viral type (isoelectric point 7), in contrast to the cellular enzyme (coded by chromosomal gene) having alkaline isoelectric point (pH 9). The results suggest that the true TK+-transformant cells are selected by the procedure used in this study.     

A cytotoxic effect associated with 9-(1,3-dihydroxy-2-propoxymethyl)-guanine is observed during the selection for drug resistant human cells containing a single herpesvirus thymidine kinase gene

A cytotoxic effect associated with 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (DHPG) was discovered while searching for spontaneous mutations in a single copy, integrated HSV-1 thymidine kinase (TK) gene in the human 143 TK- cell line. It was found that spontaneous DHPGR mutations could not be selected while other anti-TK drugs resulted in selectable mutation frequencies of 10(-4) to 10(-3). When 143 TK- cells were mixed with these HSV-1 TK+ cells and subjected to DHPG, a 90% to 100% decrease in recoverable TK- colonies was observed. In addition, the media from the HSV-1 TK+ cells metabolizing DHPG was shown to inhibit the growth of the TK- cells.     

Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus 1.

The gene for the thymidine kinase (TK) of Herpes simplex virus type 1 (HSV-1) is located in the KpnI m and BamHI p fragments of the genome (Wigler et al., Cell 11, 223-232 (1977)). These fragments have been inserted into the EcoRI and BamHI sites, respectively, of plasmid pBR322, and propagated in E.coli. The TK gene contained in the recombinant plasmids was shown to be biologically active when introduced into TK- mouse L cells. Detailed restriction site maps of the BamHI p fragment have been constructed and the approximate location of the TK gene has been determined. Mouse cells transformed with cloned HSV-1 tk+ DNA produced HSV-1-specific thymidine kinase; superinfection with HSV-1 tk- virus increased the level of TK activity tenfold, suggesting that the BamHI p sequences present in transformed cells respond to virus-encoded regulatory gene product(s).     

Influence of 6- or 8-substitution on the antiviral activity of 3-arylalkylthiomethylimidazo[1,2-a]pyridine against human cytomegalovirus (CMV) and varicella-zoster virus (VZV): part II

The synthesis of original imidazo[1,2-a]pyridines bearing a thioether side chain at the 3 position and diversely substituted on the 6 or 8 position, and their antiviral activities are reported. From the synthesized compounds, the imidazo[1,2-a]pyridines bearing a 5 membered heterocycle (thiophene, furane or pyrrole) in the 6 position or a phenylthio group in the 6 or 8 position were the most potent against human cytomegalovirus (CMV) and varicella-zoster virus (VZV), whereas several other congeners, while less potent, were more selective in their inhibitory activity against VZV and CMV. These compounds showed similar activity against thymidine kinase competent (TK+) and deficient (TK-) VZV strains, demonstrating a mechanism of action independent of the viral thymidine kinase.     

Genetic transformation of somatic cells. IV. The rate of loss of transformant phenotype depends on the structure of the transforming DNA and changes when the cells are treated with the tumor promotor 12-o-tetradecanoyl-phorbol-13-acetate

Analysis was made of the phenotype stability of some clones of thymidine kinase deficient (TK-) Chinese hamster cells transformed by thymidine kinase gene (TK-gene) of Herpes simplex virus type (HSV 1). The presence of a fragment of human satellite DNA III in the plasmid DNA carrying the TK-gene of HSV 1 reduced notably the rate of the loss of TK+-phenotype, and the treatment of the cells with a tumour promoter--12-o-tetradecanoyl-phorbol-13-acetate--immediately after transformation destabilizes TK+-phenotype of transformant clones. Removal of the eukaryotic carrier DNA for the plasmid DNA without the TK-gene of HSV 1 destabilizes the clone transformant phenotype. Changes in the structure of the plasmid DNA containing no TK-gene of HSV 1 and introduced into cells simultaneously with TK-gene containing plasmids affects the rate of the loss of TK+-phenotype transformed cells.