Inhibition of herpes simplex thymidine kinase gene expression by DNA methylation is an indirect effect (original) (raw)
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Molecular and cellular biology, 1994
In order to determine whether partial methylation of the herpes simplex virus (HSV) tk gene prevents tk gene expression, the HSV tk gene was cloned as single-stranded DNA. By in vitro second-strand DNA synthesis, specific HSV tk gene segments were methylated, and the hemimethylated DNA molecules were microinjected into thymidine kinase-negative rat2 cells. Conversion of the hemimethylated DNA into symmetrical methylated DNA and integration into the host genome occurred early after gene transfer, before the cells entered into the S phase. HSV tk gene expression was inhibited either by promoter methylation or by methylation of the coding region. Using the HindIII-SphI HSV tk DNA fragment as a primer for in vitro DNA synthesis, all cytosine residues within the coding region, from +499 to +1309, were selectively methylated. This specific methylation pattern caused inactivation of the HSV tk gene, while methylation of the cytosine residues within the nucleotide sequence from +811 to +130...
Proceedings of the National Academy of Sciences, 1987
Inhibition of herpes simplex virus (HSV) thymidine kinase (TK) gene transcription (pHSV-106, pML-BPV-TK4) by DNA methylation is an indirect effect, which occurs with a latency period of -8 hr after microinjection of the DNA into TK-rat 2 and mouse LTK-cells. We have strong evidence that chromatin formation is critical for the transition of the injected DNA from methylation insensitivity to methylation sensitivity. Chromatin was reconstituted in vitro by using methylated and mock-methylated HSV TK DNA and purified chicken histone octamers. After microinjection, the methylated chromatin was always biologically inactive, as tested by autoradiography of the cells after incubation with [3H]thymidine and by RNA dot blot analysis. However, in transformed cell lines, reactivation of the methylated chromatin occurred after treatment with 5;azacytidine. Furthermore, integration of the TK chromatin into the host genome is not required to block expression of the methylated TK gene. Mouse cells that contained the pML-BPV-TK4 chromatin permanently in an episomal state also did not support TK gene expression as long as the TK DNA remained methylated.
Molecular and cellular biology, 1988
Infection of rat embryo cells with herpes simplex virus type 2 caused undermethylation of host cell DNA synthesized during infection. DNA made prior to infection was not demethylated, but some of its degradation products, including methyl dCMP, were incorporated into viral DNA. The use of mutant virus showed that some viral DNA synthesis appears to be required for the inhibition of methylation. Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.
Nucleic Acids Research, 1980
The thymidine kinase (TK) gene of HSV-1 has been cloned in Escherichia coli K12 plasmids, pMHl, pMHlA, and pMH4. These plasmids contain a 1,920bp HSV-1 TK DNA sequence, which replaces a 2,067 bp EcoR I to Pvu II sequence of plasmid pBR322 DNA. Superhelical DNAs of plasmids pMHl, pMHlA, and pMH4 as well as plasmid DNAs cleaved by EcoR I, Hinc II, Bgl II, Sma I, and Pvu II transformed TK-deficient LM(TK-) cells to the TKe phenotype. A 1,230bp EcoR I-Sma I fragment purified from pMHl DNA (and from plasmid pAGO DNA, the parent of pMH1) also transformed LM(TKI) cells. Serological and disc PAGE studies demonstrated that the TK activity expressed in biochemically transformed cells was HSV-l-specific. The experiments suggest that the HSV-1 TK coding region may be contained within a l.lkbp DNA sequence extending from about the Hinc II (or Bgl II) cleavage site to the Sma I site. 35S-methionine labeling experiments carried out on cell lines transformed by Hinc II-cleaved pMHl DNA and by the EcoR I-Sma I fragment showed that the TKs purified from the transformed cells consisted of about 39-40,000 dalton polypeptides.
Journal of General Virology, 1983
The induction of thymidine kinase (TK) and DNA polymerase was inhibited by interferon (IFN) in mouse L-cells infected with herpes simplex virus type 1 (HSV-1). The inhibitory activity of IFN at this early stage of HSV-1 replication was followed by a reduced virus yield and was dependent on the multiplicity of infection.The expression of a cloned thymidine kinase (tk) gene of HSV-1, in biochemically transformed L-cells (LTK+), was not affected by IFN. These same LTK + cells, however, developed an antiviral state since, upon HSV-1 infection, the induction of TK and DNA polymerase of the replicating virus was inhibited by IFN. Furthermore, IFN inhibited the transactivation of the HSV-1 tk gene in the biochemicaUy transformed LTK + cells, which followed infection by a virus mutant defective in the tk gene (HSV-1 TK-). This transactivation is dependent on expression of immediate-early HSV-1 s-genes. These results indicate that IFN inhibits HSV-I replication at an early step prior to DNA synthesis. In addition, IFN displays a differential effect on the HSV-1 thymidine kinase gene, either when part of the replicating virus or when expressed as a cellular gene in biochemically transformed cells.
Journal of General Virology, 1982
We have defined the minimal size and physical map locations in the genomes of both herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) for DNA sequences capable of conferring stable biochemical transformation under thymidine kinase (TK) selection. The experiments involved transfection of Ltk-cells with either isolated virus DNA fragments or cloned pBR322 plasmids containing the 3.5 kilobase (kb) BamHI-O fragment from HSV-I(MP) or the 5.6 kb SalI-G fragment from HSV-2(333). Mapping of restriction enzyme sites within these cloned DNAs, followed by assays for colony formation in HAT medium after transfection with cleaved DNA, localized the biologically active TK-transforming sequences to lie between coordinates 0.300 and 0.313 in HSV-1 and between 0.303 and 0.315 in HSV-2. Experiments with a series of cloned plasmids containing deletions of the BamHI-O fragment towards either the 3'-or 5'-ends of the TK gene indicated that the sequences required for stable HSV-1 TK transformation lay within a 1600 base pair (bp) region at 0.303 to 0.313 map units. An internal deletion mutant plasmid, selected by a novel bacterial transfection assay for the absence of the KpnI site at 0.308, also failed to rescue Ltk-cells. With the exception of cleavage at the StuI site at 0-303 in HSV-2, which reduced activity only eightfold, all cleavages that affected TK transformation reduced the efficiency at least 50-fold. A direct comparison of the HSV-1 and HSV-2 minimal transforming regions with the nucleotide sequence of the structural HSV-1 TK gene indicates that the HSV-2 StuI site lies 30 bp beyond the poly(A) addition site at the 3'-end of TK mRNA. On the other hand, cleavage at the Sinai site in HSV-1 TK, located 80 bp in front of the poly(A) addition point, abolishes colony formation. Comparison of the putative 5'-end of the HSV-2 TK gene defined by transfection assays, with a 250 bp non-transcribed region at the front of the HSV-1 TK gene, suggests that the promoter regions contain a much higher frequency of conserved cleavage sites than do the coding portions of the two genes. Direct nucleotide sequencing of the 5'-flanking sequences for HSV-2 TK confirmed that large portions of the two promoters possess greater than 95 % sequence homology. At least 140 bp, but no more than 200 bp, of this 5'-promoter region are essential for efficient transfer and expression of the viral TK gene. Combining the results from HSV-1 and HSV-2, we conclude that a contiguous sequence of 1480 to 1540 bp is necessary to achieve at least 10 % of the maximum transformation efficiency.
Hemimethylation of DNA prevents chromatin expression
Proceedings of the National Academy of Sciences, 1990
The activity of hemimethylated herpes simplex virus thymidine kinase DNA and chromatin was analyzed by microin'ection and thymidine incorporation into the DNA of thymidine kinase-negative Rat2 cells. Hemimethylated DNA was obtained by in vitro replication of single-stranded M13 DNA constructs and of chromatin produced by in vitro reconstitution of the DNA with purified chicken histone octamers. We found that methylation of either the coding or the noncoding DNA strand was sufficient to block expression of the hemimethylated chromatin. In contrast, the hemimethylated DNA was as active as the unmethylated control DNA after micro'n ection until chromatin formation occurred in the recipient cells. Microinjection of chromatin hemimethylated by bacterial Hae Ill methyltransferase excluded the possibility that inactivation was caused by symmetrical methylation of the injected molecules.
Proceedings of the National Academy of Sciences, 1977
Transformation of human cells from a thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75)-negative to a thymidine kinase-positive phenotype has been achieved by using purified DNA from herpes simplex virus type 2. The specific activity of the DNA was in the range 0.5 to 2.0 transformants per microng and the efficiency of gene transfer was up to 1 transformant per 10(5) recipient cells. Several transformed lines able to grow continuously in medium selective for thymidine kinase-positive cells have been established. All of these lines express a thymidine kinase activity of viral origin but they differ from each other in the stability of enzyme expression. Subclones derived from a given transformed line inherited the degree of stability of the parental line.