Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus (original) (raw)
Related papers
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...
Nucleic Acids Research, 1985
The biological activity of in vitro methylated HSV-TK DNA was analysed after microinjection into thymidine kinase negative rat 2 cells. It was found that the fully methylated DNA (HpaII methylase) was as active as the non methylated control DNA for about 48 hours after injection. DNA reextraction experiments and blot analysis showed that DNA demethylation was not the reason for the observed TK activity. With prolonged cultivation time the methylated DNA becomes rapidly inactive and 100 hrs after injection thymidine incorporation was no longer detectable in the recipient cells. In transformed cells, obtained after coinjection with SV40 DNA, the HSV-DNA was partially demethylated and inactive. Addition of 5-azacytidine to the culture medium induced further demethylation and reactivation of the thymidine kinase gene.
Synthesis of viral and host DNA in isolated chromatin from herpes simplex virus-infected HeLa cells
Journal of Virology, 1978
DNA synthesis in chromatin isolated from herpes simplex virus type 1-infected HeLa cells (HSV chromatin) was examined in vitro. The HSV chromatin. was found to carry out an initial limited synthesis of DNA in vitro, 50 to 64 pmol of dTMP incorporated in 106 nuclei per 10 min, which is comparable to that found in nuclei isolated from HSV-infected cells. DNA synthesis in vitro proceeded for only 30 min, and both HSV DNA and host DNA were synthesized in significant amounts. The HSV and host DNA syntheses in isolated chromatin were inhibited to the same extent by anti-HSV antiserum or by phosphonoacetic acid. The results indicate that the HSV-induced DNA polymerase is most likely involved in the synthesis of host and HSV DNA in isolated chromatin, even though this chromatin contains small amounts of the host-y-polymerase in addition to the HSV-induced DNA polymerase. The HSV chromatin contains no detectable levels of DNA polymerases a and f, even though infected cells have normal, or increased, levels of these enzymes. Nuclei isolated from uninfected and virus-in-chromatin preparation isolated from the nuclei fected eucaryotic cells have been used to study of HSV-infected HeLa cells can also replicate in vitro DNA replication. It has been demon-both HSV and host DNA and that the HSVstrated that DNA synthesis by isolated nuclei induced DNA polymerase is responsible for both requires deoxynucleotides, ATP, and cytoplas-the HSV DNA and the host DNA syntheses in mic fraction (see review [10]). Although DNA this system. synthesis by isolated nuclei produces some abnormal distribution of DNA sequences (32), the MATERIALS AND METHODS nuclei system has been used successfully to de-Radiochemicals. [Methyl-3H]dTTP was purtect stimulatory factors in the cytoplasm (4, 11) chased from Schwarz/Mann, and [methyl-3H]thymiand to show the involvement of RNA in DNA dine, [a-3P]dTTP and [5-'5I]dCTP were obtained replication (22, 23, 27, 28, 31). from New England Nuclear Corp. Recently, the extraction and reconstitution of Chemicals. Deoxynucleoside triphosphates and rinuclear DNA synthesis in isolated mammalian bonucleoside triphosphates were purchased from Sigma Chemical Co. Bromodeoxyuridine triphosphate cell nucei has been attempted (26), and parteal was obtained from P-L Biochemicals, and Sarkosyl separation of a putative adenovirus DNA repli-NL-97 was from the Ciba-Geigy Corp. Phosphonoacecation complex from adenovirus-infected nuclei tic acid was a gift of E. Heimer and A. Cook from the (33) has been suggested. The finding that simian Hoffman-La Roche Research Laboratories. Pancreatic virus 40 DNA synthesis can occur in a nucleo-DNase I and RNase were from Worthington Biochemprotein complex (9, 25) and that HeLa DNA ical Corp., and RNase Ti and Pronase were from synthesis is observed in isolated chromatin (12) Calbiochem. HSV antiserum was the same as dehas shown that chromatin is active for DNA scribed previously (7). Ethyleneglycol-bis(fi-aminosynthesis in vitro. Chromatin may offer advan-ethyl ether)-NN-tetraacetic acid (EGTA) was obtages over the isolad ntained from Sigma Chemical Co. tages over the Isolated nuclei in itS accessiblity Cell culture and virus infection. HeLa F cells to DNA polymerases or other enzymatic factors were used for the proliferation of HSV type 1 (HSV-1) (12,15). (Miyama strain) (20). HeLa F cells were grown in Previously, a number of groups have reported monolayer culture in a glass roller bottle (10 by 50 cm) that nuclei from herpes simplex virus (HSV)or in 150-cm2 culture flasks with F-li medium (Grand infected cells could synthesize HSV DNA in Island Biological Co.) supplemented with 10% fetal vitro (2, 6, 7, 14). This paper will show that a calf serum, 4 mM glutamine, and 1% streptomycinpenicillin. When the cells approached confluency, they
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.
Journal of Virology, 1982
The nature of Moloney murine leukemia virus (M-MuLV)-specific proviral DNA in exogenously infected mouse cells was studied. M-MuLV clone A9 cells, NIH-3T3 fibroblasts productively infected with M-MuLV, were used. These cells contain 10 to 15 copies of M-MuLV proviral DNA. The state of methylation of M-MuLV proviral DNA was examined by cleaving A9 cell DNA with restriction endonucleases which have the dinucleotide CpG in their cleavage sequences. Analysis with such enzymes, which recognized nine different sites in M-MuLV DNA, indicated that most if not all of the M-MuLV proviruses in A9 cells were completely unmethylated. An individual proviral integration was examined, using as probe adjacent single-copy cellular sequences. These sequences were obtained from a lambda phage recombinant clone containing an M-MuLV provirus from the A9 cells. This individual integration also showed no detectable methylation. In contrast, endogenous MuLV-related sequences present in NIH-3T3 cells before ...
Methylation of foreign DNA sequences in eukaryotic cells
Proceedings of the National Academy of Sciences, 1980
The herpesvirus thymidine kinase gene has been used to introduce foreign DNA sequences into mouse L cells by DNA-mediated gene transfer. These inserted genes were then assayed for methylation at the specific sequence C-C-G-G by using the restriction enzyme isoschizomers Hpa II and Msp I. Despite the fact that 70% of the cellular C-C-G-G sites are methylated, herpesvirus sequences, plasmid DNA, and growth hormone gene DNA were found to remain unmethylated in 90% of the clones that contain these genes. DNA that had been methylated in vitro with Hpa II methylase was also inserted into L cells. The presence of this modification in the vector DNA did not, however, guarantee that these sequences remained methylated in the recipient clones. Only 10% of all transformed clones were found to contain methylated C-C-G-G sequences in the vector DNA, and these modifications were stable for 25-50 generations. Hha I and Mbo I were used to probe for methyl groups at these restriction sites, but none...
Histone H3 Lysine 4 Methylation Marks Postreplicative Human Cytomegalovirus Chromatin
Journal of Virology, 2012
In the nuclei of permissive cells, human Cytomegalovirus genomes form nucleosomal 26 structures initially resembling heterochromatin but gradually switching to a euchromatin-like 27 state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked 28 increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used 29 ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine 30 the impact of DNA replication on histone modification dynamics at the viral chromatin. The 31 changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-32 independent fashion. In contrast, the increase in H3 K4 methylation proved to widely depend on 33 viral DNA synthesis. Consistently, labeling of nascent DNA using "click chemistry" revealed 34 preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or 35
Molecular and cellular biology, 1983
The transforming activity of cloned Moloney sarcoma virus (MSV) proviral DNA was inhibited by in vitro methylation of the DNA at cytosine residues, using HpaII and HhaI methylases before transfection into NIH 3T3 cells. The inhibition of transforming activity due to HpaII methylation was reversed by treatment of the transfected cells with 5-azacytidine, a specific inhibitor of methylation. Analysis of the genomic DNA from the transformed cells which resulted from the transfection of methylated MSV DNA revealed that the integrated MSV proviral DNA was sensitive to HpaII digestion in all cell lines examined, suggesting that loss of methyl groups was necessary for transformation. When cells were infected with Moloney murine leukemia virus at various times after transfection with methylated MSV DNA, the amount of transforming virus produced indicated that the loss of methyl groups occurred within 24 h. Methylation of MSV DNA at HhaI sites was as inhibitory to transforming activity as me...
Virology, 2008
Human papillomavirus-16 (HPV-16) genomes in cell culture and in situ are affected by polymorphic methylation patterns, which can repress the viral transcription. In order to understand some of the underlying mechanisms, we investigated changes of the methylation of HPV-16 DNA in cell cultures in response to cellular differentiation, to recombination with cellular DNA, and to an inhibitor of methylation. Undifferentiated W12E cells, derived from a precancerous lesion, contained extrachromosomal HPV-16 DNA with a sporadically methylated enhancer-promoter segment. Upon W12E cell differentiation, the viral DNA was demethylated, suggesting a link between differentiation and the epigenetic state of HPV-16 DNA. The viral genomes present in two W12I clones, in which individual copies of the HPV-16 genome have integrated into cellular DNA (type 1 integrants), were unmethylated, akin to that seen in the cervical carcinoma cell line SiHa (also a type 1 integrant). This finding is consistent with hypomethylation being necessary for continued viral gene expression. In contrast, two of three type 2 integrant W12I clones, containing concatemers of HPV-16 genomes integrated into the cellular DNA contained hypermethylated viral DNA, as observed in the cervical carcinoma cell line CaSki (also a type 2 integrant). A third, type 2, W12I clone, interestingly with fewer copies of the viral genome, contained unmethylated HPV-16 genomes. Epithelial differentiation of W12I clones did not lead to demethylation of chromosomally integrated viral genomes as was seen for extrachromosmal HPV-16 DNA in W12E clones. Hypomethylation of CaSki cells in the presence of the DNA methylation inhibitor 5-aza-2'-deoxycytidine reduced the cellular viability, possibly as a consequence of toxic effects of an excess of HPV-16 gene products. Our data support a model wherein (i) the DNA methylation state of extrachromosomal HPV16 replicons and epithelial differentiation are inversely coupled during the viral life cycle, (ii) integration of the viral genome into the host chromosome events leads to an alteration in methylation patterns on the viral genome that is dependent upon the type of integration event and possibly copy number, and (iii) integration universally results in the viral DNA becoming refractory to changes in methylation state upon cellular differentiation that are observed with extrachromosomal HPV-16 genomes.