Nucleotide sequence of the herpes simplex virus type 2 thymidine kinase gene.

We have determined the complete nucleotide sequence of the thymidine kinase gene of herpes simplex virus (HSV) type 2 strain 333. The sequence of the thymidine kinase gene exhibits an open translational reading frame of 1,128 nucleotides encoding a protein of 376 amino acids. The DNA sequence was compared with that of the HSV type 1 thymidine kinase gene from strain MP (S. L. McKnight, Nucleic Acids Res. 8:5949-5964, 1980) and from strain CL 101 (M. J. Wagner, J. A. Sharp, and W. C. Summers, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981) to assess the extent of intra- and intertypic variation for one viral gene. The nucleotides encoding the structural gene varied 1.7% between the two HSV type 1 strains and 19% between HSV type 1 and HSV type 2, which translated to differences in the amino acid sequence of the two proteins of 1.9 and 27%, respectively. The DNA encoding the 5' regulatory sequences appeared to be more conserved than the DNA coding for the structural gene, and the DNA at the 3' end of the gene was the least homologous.     

Initiation of herpes simplex virus thymidine kinase polypeptides.

When employed as a transgene reporter, the herpes simplex type 1 virus (HSV1) thymidine kinase gene (tk) is ectopically expressed in mouse testis. The principal testicular mRNA lacks the 5'-end of the tk reading frame. As a result the principal translation products, P2 and P3, are N-terminally truncated. These co-migrate in SDS-PAGE with polypeptides synthesised during HSV1 infection that were previously thought to be initiated at methionine codons ATG46 and ATG60. Prompted by these observations we generated modified tk genes each carrying only one of the first three ATG codons. Transfected cells expressed both full-length enzyme (P1) and P2 when only ATG1 was unmodified, P2 and P3 when only ATG46 was unmodified or P2 and a fourth polypeptide (P4) when only ATG60 was unmodified. Our observations indicate that P3 is initiated at ATG46 rather than ATG60, while P2 is initiated at a non-ATG codon rather than ATG46 and P4 is initiated at ATG60. When either of two putative non-ATG initiation codons was modified P2 was no longer produced. Cells mainly expressing either P1 or P3 exhibited the same sensitivity to Ganciclovir as cells transfected with the unaltered tk gene. P1 and P3 both have TK activity while P4 probably has none.     

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.     

Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro

The herpes simplex 1 virus thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and for imaging intracellular molecular events and cell trafficking in living subjects. Two in vitro methods are available to assay gene expression of HSV1-tk or HSV1-sr39tk in cells or tissues. One method determines the level of HSV1-TK or HSV1-sr39TK enzyme activity in cell or tissue lysates by measuring the amount of the radiolabeled substrates that have been phosphorylated by these enzymes in a fixed amount of cell lysate protein after a fixed incubation time. The other method, called the 'cell-uptake assay', takes into account the natural uptake and efflux characteristics of the radiolabeled substrate by specific cells, in addition to the level of HSV1-TK or HSV1-sr39TK activity. Both of these assays can be used to validate molecular models in cultured cells, prior to studying them in living research subjects. Each of these assays can be completed in one day.     

DNA-mediated gene transfer in Friend leukemia cells by cotransfection of simian virus 40 DNA with herpes simplex virus thymidine kinase DNA.

Thymidine kinase-negative Friend leukemia cells were cotransfected with simian virus 40 (SV40) DNA and thymidine kinase gene DNA of herpes simplex virus type 1. The transfected thymidine kinase-positive cells were selected in HAT medium, and SV40 T-antigen expression was observed over many months in cells cultured under selective conditions, and after adaptation to normal growth medium under nonselective conditions. It was shown by Southern blot hybridization that SV40 DNA was integrated in multiple copies in the chromosomal DNA of several clones. All SV40 DNA-containing Friend leukemia cell clones analyzed were able to undergo induced erythroid differentiation. Induced cultures still expressed SV40 T-antigen to the same extent that untreated control cultures did.     

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.     

Conversion of Friend mink cell focus-forming virus to Friend spleen focus-forming virus by modification of the 3'' half of the env gene.

The 3' half of the env gene of the dualtropic Friend mink cell focus-forming virus was modified by replacing the restriction enzyme fragment of the genome DNA with the corresponding fragment of the acutely leukemogenic, polycythemia-inducing strain of Friend spleen focus-forming virus (F-SFFVP) genome DNA. Replacement with the fragment of F-SFFVP env containing the 585-bp deletion, the 6-bp duplication, and the single-base insertion converted the resulting chimeric genome so that the mutant had a pathogenic activity like that of F-SFFVP. Replacement with the fragment containing only the 585-bp deletion did not result in a pathogenic virus. However, when this virus pseudotyped by Friend murine leukemia virus was passaged in newborn DBA/2 mice, we could recover weakly pathogenic viruses with a high frequency. Molecular analysis of the genome of the recovered virus revealed the presence of a single-base insertion in the same T5 stretch where the wild-type F-SFFV env has the single-base insertion. These results provided evidence that the unique genomic structures present in the 3' half of F-SFFV env are the sole determinants that distinguish the pathogenicity of F-SFFV from that of Friend mink cell focus-forming virus. The importance of the dualtropic env-specific sequence present in the 5' half of F-SFFV env for the pathogenic activity was evaluated by constructing a mutant F-SFFV genome in which this sequence was replaced by the ecotropic env sequence of Friend murine leukemia virus and by examining its pathogenicity. The results indicated that the dualtropic env-specific sequence was essential to pathogenic activity.     

Transfection of mouse cells with thymidine kinase gene of herpes simplex virus

A mouse cell line (LP1-1) was established from the murine L cells deficient in thymidine kinase (L-M(TK ^–)) by prolonged selective culture on the hypoxanthine-aminopterine-thymidine (HAT) medium following transfection with the thymidine kinase gene of herpes simplex virus type-I (HSVTK). Southern blot analysis has shown that the viral TK gene was integrated into one of the chromosomal loci by a single copy. From this established cell line, the 5-bromo-2-deoxyuridine (BrdU) resistant revertant was brought out at a frequency of 1×10^–6 and from these BrdU resistant revertants (LP1BU), one out of 1×10⁵ cells could return to the HAT-resistant phenotype. The established LP1-1 cell line showed a typical biphasic nature of DNA synthesis as determined by the ³H-thymidine incorporation test. The activity of thymidine kinase was shown to be equivalent to that of the DNA polymerase- when the whole nuclear fraction or the nuclear matrix were used for examination. These results indicate that the transfected viral TK gene can be expressed under the normal cell-cycle regulation and its gene product can act as a component of the multienzyme complex which is responsible for DNA replication.     

Role of the viral and cellular encoded thymidine kinase in the repair of UV-irradiated herpes simplex virus

A strain of herpes simplex type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) gene and a thymidine kinase deficient (tk-) mutant strain (HSV-1:PTK3B) were used as probes to examine the repair of UV-damaged viral DNA in one tk- (143) and two tk+ (R970-5 and AC4) human cell lines. UV survival for each HSV-1 strain was similar for infection of both tk- and tk+ cells suggesting that the repair of viral DNA was not dependent on the expression of a functional cellular tk gene. In contrast, UV survival of HSV-1:PTK3B was substantially reduced compared to HSV-1:KOS when infecting all 3 human cell lines, as well as Vero monkey kidney cells and LPM1A mouse cells. These results suggest that the repair of UV-irradiated HSV-1 in lytically infected mammalian cells depends, in part at least, on the expression of the viral encoded tk.     

The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene

This paper presents the nucleotide sequence of the Herpes Simplex Virus thymidine kinase (tk) gene. The position on the DNA sequence corresponding to the 5' and 3' termini of tk messenger RNA have been mapped. The mRNA termini are separated by slightly more than 1,300 nucleotides. The same 2,300 nucleotide segment of tk coding strand DNA is fully protected from S1 nuclease digestion when hybridized to tk mRNA. The location and size of the mRNA-coding segment corresponds to a region of the viral DNA that is essential for tk gene expression in microinjected frog oocytes. The nucleotide sequence of the HSV tk gene exhibits an open translational reading frame of 376 codons that extends from the methionine codon most proximal to the 5' terminus of tk mRNA to a UGA stop codon approximately 70 nucleotides from the poly-A addition site. The results of these experiments indicate that the tk gene is not interrupted by intervening DNA sequences, and that certain oligonucleotide sequences adjacent to the termini of the tk gene are homologous to similarly positioned sequences common to structural genes of eukaryotic cells.     

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.     

Control of the expression of a herpes simplex virus thymidine kinase gene incorporated into thymidine kinase-deficient mouse cells.

When thymidine kinase-deficient mouse cells "transformed" by in activated herpes simplex virus and expressing the viral thymidine kinase (TK) are grown in nonselective medium, there is an exponential decay in the proportion of cells that continue to express the viral enzyme. However, the viral TK can be reactivated at a frequency of approximately 1 cell in 10(6) in every population that has lost TK activity. When cells in which the viral TK has been reactivated are grown in nonselective medium, a decay in the expression of the viral enzyme occurs again at the same rate as in the initial transformed population. Studies on the reactivation of viral TK indicate that reappearance of the enzyme is not induced by the selective medium (HAT) used to detect cells in which the enzyme has reappeared. Furthermore, treatments known to induce latent viruses in other systems--eg, exposure of the cells to mutagens or cell fusion--do not affect the frequency with which viral TK is reactivated.     

Integration of spleen focus-forming virus proviruses in Friend tumor cells.

Using the Southern blot procedure, we studied the presumed spleen focus-forming virus (SFFV) provirus integration sites in the genome of the premalignant and the malignant cells isolated during the course of Friend erythroleukemia. Two restriction endonucleases, PstI and BamHI, discriminated the presumed integrated SFFV proviruses from the endogenous xenotropic-mink cell focus-forming viral sequences. No SFFV integration sites were detectable in the premalignant cells, suggesting a random integration of SFFV proviruses in the genome of these cells. In contrast, SFFV proviruses were detected at a single or very few sites in the genome of all malignant cells we analyzed. These results indicate that the event leading to the malignant transformation in acute Friend leukemia is clonal. In two of the six animals examined, tumors cells isolated from the spleens and the livers of individual mice showed identical SFFV integration patterns. This last result suggests that in some cases different tumors in a same leukemic animal could be derived from a unique clonal event.     

3'-Amino thymidine affinity matrix for the purification of herpes simplex virus thymidine kinase.

A simple procedure for preparation of an affinity resin with 3''-amino thymidine linked to the carboxyl residues on 6-amino-hexanoic agarose is described. We have used this column for a rapid and simple purification of the thymidine kinase encoded by the herpes simplex virus type 1 genome. This resin has two major advantages over the most widely use used resin made with thymidine-p-nitrophenyl phosphate: first it is easily obtainable, and second, it is not subject to destruction by phosphodiesterases. The two resins are very similar in behavior and the resin made with amino thymidine has allowed us to prepare large quantities of highly purified HSV TK for crystallization studies.     

The herpes simplex virus thymidine kinase gene as a conditional negative-selection marker gene in Arabidopsis thaliana.

The human herpes simplex virus thymidine kinase type 1 gene (HSVtk) acts as a conditional lethal marker in mammalian cells. The HSVtk-encoded enzyme is able to phosphorylate certain nucleoside analogs (e.g. ganciclovir, an antiherpetic drug), thus converting them to toxic DNA replication inhibitors. The utility of HSVtk as a conditional negative-selection marker was explored in Arabidopsis thaliana (L.) Heynh. HSVtk was introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic plants were morphologically indistinguishable from wild type and exhibited normal fertility. Ganciclovir at 10(-5) to 10(-4) M drastically reduced shoot regeneration on transgenic, HSVtk+ root explants or callus formation on HSVtk+ leaf explants but did not affect the wild-type cultures. There was a 35-fold reduction in shoot regeneration 8 d after transfer to shoot-induction medium. Negative selection against HSVtk activity along with kanamycin selection was also efficient in Agrobacterium-mediated gene transfer experiments. Shoot regeneration was 25 times lower on double-selective (ganciclovir plus kanamycin) plates than in the kanamycin control. This regeneration rate in double-selective plates is in the range of the frequency of shoots normally escaping kanamycin selection in Arabidopsis cultures.     

Expression of herpes simplex virus thymidine kinase gene in aquatic filamentous fungus Achlya ambisexualis

We have constructed a plasmid vector pSV2neo-MK alpha G in which the structural tk gene for Herpes simplex virus thymidine kinase (HSV-TK) was placed downstream from the metallothionein-I promoter. The vector also contained the selection marker aminoglycoside 3'-phosphotransferase (Km). This vector was able to transform the filamentous fungus Achlya ambisexualis and G-418-resistant colonies were obtained. Southern blot analyses revealed that multiple bands hybridizing to the HSV tk gene probe were present in the genomic DNA of the transformants. Upon analysis by gel electrophoresis, one of the transformants exhibited TK activity bearing electrophoretic mobility similar to that of the HSV-TK. An increase of approx. 40% of [3H]thymidine uptake and incorporation into cellular DNA was also observed in this transformant. This study suggested that the HSV tk gene can be expressed in the fungus A. ambisexualis that can be considered as a candidate host cell for further gene-expression studies.     

Heteroduplex analysis of molecular clones of the pathogenic Friend virus complex: Friend murine leukemia virus, Friend mink cell focus-forming virus, and the polycythemia- and anemia-inducing strains of Friend spleen focus-forming virus.

The pathogenic Friend virus complex is of considerable interest in that, although members of this group are genetically related, they differ markedly in biochemical and biological properties. Heteroduplex mapping of molecular clones of the Friend virus complex, which includes the replication-competent ecotropic Friend murine leukemia virus (F-MuLV) and mink cell focus-forming virus (F-MCF) and replication-defective polycythemia- and anemia-inducing strains of spleen focus-forming virus (SFFVp and SFFVa, respectively), was employed to provide insight into the molecular basis of their relationships. In heteroduplexes of F-MuLV X F-MCF, a major substitution of 0.89 kilobases in the env gene of F-MCF was discerned. Heteroduplexes of SFFVp X F-MuLV or F-MCF and SFFVa X F-MuLV or F-MCF showed several major deletions in the pol gene region and a single major deletion in the 3' half of the env gene region of SFFVp and SFFVa. A major substitution of 0.89 kilobases was mapped to the 5' end of the env deletion of SFFVp and SFFVa in heteroduplexes with F-MuLV, similar to that seen in F-MuLV X F-MCF heteroduplexes. In contrast, this env gene region was totally homologous in F-MCF X SFFVp or SFFVa and SFFVp X SFFVa heteroduplexes. Our results suggest that (i) both SFFVp and SFFVa lack part of the env gene at its 3' end, corresponding to the p15(E) coding region, (ii) major deletions occur in the pol and env genes which account for the replication defectiveness of SFFVp and SFFVa, (iii) minor substitutions occur in the gag gene region of SFFVa that are not present in SFFVp, F-MuLV, or F-MCF, (iv) a major substitution exists in the gp70 region of the env gene between F-MuLV and F-MCF that probably accounts for the differences in their host range specificities, (v) this substitution in F-MCF is identical to the gp70 part of the gp52 coding region of SFFVp and SFFVa, and (vi) heteroduplexes to F-MCF show unambiguously that no additional large substitutions are present in SFFVp or SFFVa that could account for differences in their leukemogenicity.