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.     

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.     

Replication and virulence of pseudorabies virus mutants lacking glycoprotein gX.

Pseudorabies virus (PRV) glycoprotein gX accumulates in the medium of infected cells. In an attempt to study the function of gX, two viruses were constructed that lacked a functional gX gene. One virus, PRV delta GX1, was derived by insertion of the herpes simplex virus thymidine kinase gene into the gX-coding region. The other virus, PRV delta GXTK-, was derived by subsequent deletion of the inserted herpes simplex virus thymidine kinase gene. Both viruses replicated in cell cultures but produced no gX. Furthermore, PRV delta GX1 was capable of killing mice with a 50% lethal dose of less than 100 PFU.     

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.     

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.     

Expression from an internal AUG codon of herpes simplex thymidine kinase gene inserted in a retrovirus vector.

We identified structural features that affect the expression of an exogenous gene inserted into a retrovirus vector constructed by using spleen necrosis virus, an avian retrovirus. The thymidine kinase gene from herpes simplex virus type 1 containing deletions in the promoter and terminal sequences of the mRNA was inserted into spleen necrosis virus. We found that synthesis of thymidine kinase by the recovered virus was apparently initiated from internal AUG residues. At least in some cases, however, the level of expression depended on the number of AUGs and the nucleotide sequence around the AUGs that preceded the initiator codon of the thymidine kinase gene.     

Functional suppression in mammalian cells of nonsense mutations in the herpes simplex virus thymidine kinase gene by suppressor tRNA genes.

A nonsense mutation (UAG) in the thymidine kinase gene of herpes simplex virus type 1 can be suppressed in vivo to produce active thymidine kinase by prior infection with a defective simian virus 40 stock which acts as a vector to introduce a functional suppressor tRNA gene into mammalian cells in culture. The suppression is specific for UAG, but not UGA or missense, mutants and restores thymidine kinase activity to 20 to 40% of the wild-type level. These results suggest that many cell lines susceptible to simian virus 40 infection may be transiently converted to a suppressor-positive phenotype for use in the genetic study of mammalian viruses.     

Immunity in the female genital tract after intravaginal vaccination of mice with an attenuated strain of herpes simplex virus type 2.

Herpes simplex virus type 2 is a common human venereal pathogen which causes lethal neurological illness after intravaginal inoculation into BALB/cJ mice. To investigate whether an attenuated, nonlethal strain of this virus would confer immunity after inoculation of mice, we constructed a strain containing a partial deletion of the thymidine kinase gene, which is necessary for viral replication and spread in sensory ganglia. Unlike its wild-type counterpart, this deletion-containing strain of herpes simplex virus type 2 caused mild clinical disease and was not lethal when studied in an age-dependent murine model of intravaginal infection. Furthermore, after intravaginal infection, the deletion-containing strain could not be isolated from sensory ganglia at a time when wild-type virus was abundant. Of greater significance, intravaginal inoculation with the deletion-containing strain rendered mice completely resistant to rechallenge with a 10-fold 50% lethal dose of wild-type virus. These results suggest that a strain of herpes simplex virus type 2 containing a deletion of the thymidine kinase gene will be useful in studying the cellular basis of mucosal immunity in the genital tract.     

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.     

Introduction of the herpes simplex virus thymidine kinase gene into mouse cells using virus DNA or transformed cell DNA.

Cells lacking the enzyme thymidine kinase (LMTK- cells) have been transformed to a kinase-positive phenotype using sheared herpes simplex virus (HSV) DNA, and the enzyme found in these transformed cells is HSV-specific. One of the cell lines is able to complement the functional defect found in two temperature-sensitive mutants of HSV 1, and reversion of the cells to a thymidine kinase-negative phenotype results in the loss of this capability. The HSV thymidine kinase gene can also be introduced into LMTK- cells using DNA extracted from transformed cells, and the high efficiency of this procedure suggests that the state of the virus DNA in transformed cells is different from that of DNA in virus particles.     

Molecular biological study of the vaccinia virus genome. IV. The late nonstructural 36K protein of vaccinia virus is vitally important

Two genetics markers: the herpes simplex virus thymidine kinase and Escherichia coli beta-galactosidase genes were inserted into the 36K protein gene of vaccinia virus located in a HindIII-P DNA fragment. An unstability of recombinant viruses with Lac(+)-phenotype were discovered. A mechanism of viruses unstable variants formation was proposed, it was confirmed by the results of hybridisation analyses of virus recombinant genomes. The importance of a late nonstructural 36K protein gene for virus reproduction was demonstrated.     

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.     

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.     

The effect of cell synchronization on the efficiency of stable gene transfer by electroporation

We synchronized thymidine kinase deficient mouse Ltk-cells by two different methods, hydroxyurea double-block treatment or aphidicolin single-block treatment and transformed them with the cloned herpes simplex virus thymidine kinase gene at various time intervals by the electroporation technique. Marked enhancement of stable transformation efficiency was observed at the time corresponding to the peak of G2/M phase. These results suggest that the G2/M phase is the most efficient period for stable gene transfer by electroporation.     

Drosophila P element-enhanced transfection in mammalian cells.

We constructed a gene transfer vector containing the herpes simplex virus type 1 thymidine kinase (TK) gene flanked by Drosophila P element terminal repeats (W. R. Engels, Annu. Rev. Genet. 17:315-344). This vector was introduced into mouse LTK- cells and enhanced the frequency of stable transformation to the TK+ phenotype by approximately 50-fold relative to a similar plasmid lacking the P element terminal repeats.     

Dominant positive and negative selection using a hygromycin phosphotransferase-thymidine kinase fusion gene.

The hygromycin phosphotransferase gene was fused in-frame with the herpes simplex virus type 1 thymidine kinase gene. The resulting fusion gene (termed HyTK) confers hygromycin B resistance for dominant positive selection and ganciclovir sensitivity for negative selection and provides a means by which these selectable phenotypes may be expressed and regulated as a single genetic entity.