Induction of cellular DNA polymerases in a Burkitt lymphoma-derived cell line by treatment with 5-iododeoxyuridine

Cellular DNA polymerases of a Burkitt lymphoma-derived cell line (P3HR-1) were found to be greatly induced by treatment of the cells with 5-iododeoxyuridine (IUdR) at a concentration which induces Epstein-Barr virus (EBV) early antigen (EA) expression. The activities of all the DNA Polymerases alpha, beta and gamma in P3HR-1 cells increased 7-9 fold by exposure of the cells to IUdR (25 micrograms/ml) for 3 days, while the EBV-coded DNA polymerase activity in the cell remained undetectable under the assay conditions employed. Under the same culture conditions with IUdR, EA-positive P3HR-1 cells increased to 16.6% which was much higher than that of the non-treated control cells (0.32%). On the other hand, another Burkitt lymphoma cell line, Raji, had very low incidence (1.27%) of EA induction by IUdR-treatment and the level of DNA polymerase activities remained almost unchanged. From these results it seems that the increase in DNA polymerase activity during the treatment of P3HR-1 cells with IUdR is closely related to high incidence of EA expression in these Burkitt lymphoma cells. Also, the finding has revealed yet unknown effect of IUdR on cultured cells and provides a useful tool to obtain a large quantity of the induced cellular DNA polymerases from the P3HR-1 and KB cells.     

巴豆油正丁酸对Raji细胞的联合诱导作用

以4mM正丁酸,400ng/ml巴豆油对Raji细胞进行联合激发,24小时后DNase活性及EA阳性细胞百分率均平行地急剧上升,72小时后达到高峰,以后EA阳性细胞急速下降,而DNase活性仍保持在高峰水平,粗酶液中的DNase活性能被EBV阳性血清中和90％以上。 激发Raji细胞粗酶液DEAE层析谱显示,有三个DNase活性峰,主峰洗脱浓度为220mM磷酸盐。检测了未激发及经激发(48小时)Raji细胞提取液的DNA多聚酶活性,前者能被50mM(NH_4)_2SO_4所抑制,抑制?原酶活性的23.3％,后者能被50mM(NH_4)_2SO_4所激活,激活为原酶活性的147.7％。     

Epstein-Barr virus induces fragmentation of chromosomal DNA during lytic infection.

Pulsed-field agarose gel electrophoresis showed that fragmentation of chromosomal DNA in Raji cells was induced by infection with the P3HR-1 strain of Epstein-Barr virus (EBV). S1 nuclease treatment of the agarose plugs containing cells suggested that the majority of DNA fragments did not contain single-strand gaps. Chromosomal DNA fragmentation was inhibited by cycloheximide, indicating that protein synthesis was required for DNA fragmentation. Phosphonoacetic acid, an inhibitor of EBV DNA polymerase, did not inhibit fragmentation of chromosomal DNA. These findings suggest that EBV-specific early proteins participate in fragmentation of chromosomal DNA. Chromosomal DNA of P3HR-1 cells was also fragmented by treatment with n-butyrate plus 12-O-tetradecanoylphorbol-13-acetate (TPA), which induced activation of latent EBV genome following viral replication. In addition, fragmentation of DNA preceded cell death during lytic infection. These results suggest that fragmentation of chromosomal DNA is generally induced during EBV replication and probably contributes to the cytopathic effect of EBV. The role of DNA fragmentation in death of infected cells is discussed in relation to apoptosis.     

Effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr virus DNA replication.

The effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr virus (EBV) DNA replication in the lymphoblastoid cell lines P3HR-1 and Raji is reported. Acyclovir at a concentration of 100 microM completely inhibited EBV DNA synthesis in superinfected Raji cells, but did not inhibit DNA synthesis in mock-infected cells. The number of EBV genome equivalents per cell in the virus-producing cell line P3HR-1 was significantly reduced by acyclovir, whereas the number of latent EBV genomes in Raji cells was not affected by the drug. In situ cytohybridization performed on untreated P3HR-1 cultures revealed the presence of relatively large amounts of EBV DNA in 15 to 20% of the cells. After a 100 microM drug treatment, no P3HR-1 cells contained levels of EBV DNA detectable by in situ cytohybridization. Indirect immunofluorescence studies demonstrated that during treatment with 100 microM acyclovir for 7 days, the percentage of P3HR-1 cells expressing viral capsid antigen was reduced. The EBV DNA remaining in P3HR-1 cells after treatment with 100 microM acyclovir (approximately 14 genomes per cell) had the properties of covalently closed circular DNA with an average molecular weight of 108 X 10(6), as determined by contour length measurements.     

The circular intracellular form of Epstein-Barr virus DNA is amplified by the virus-associated DNA polymerase.

Selective DNA extraction and hybridization procedures were used to estimate the relative number of covalently closed circular viral genomes in cultures of Epstein-Barr virus (EBV)-transformed cells. In virus-producing P3HR-1 cultures that were exposed for 11 days to phosphonoacetic acid or to acyclovir, the content of covalently closed circular EBV DNA was reduced ca. 70% relative to a control culture without drug. The EBV plasmid content of Raji, a virus nonproducer cell line, was not reduced by exposure to these compounds. When P3HR-1 cultures were exposed to 12-O-tetradecanoylphorbol-13-acetate, the number of circular genomes per cell increased. These findings indicate that two enzyme activities synthesize circular EBV DNA and that the virus-associated DNA polymerase synthesizes most of the circular EBV DNA in a virus producer culture. It is suggested that the circular genomes synthesized by the viral enzyme are intermediates in the syntheses of linear virus DNA.     

Amplification of Epstein-Barr virus (EBV) DNA by superinfection with a strain of EBV derived from nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) from a nasopharyngeal carcinoma (NPC) hybrid cell line (NPC-KT) lacking defective viral DNA molecules superinfected Raji cells and induced EBV early antigens (EA), as did virus from P3HR-1 cells, which contained defective molecules. The EBV polypeptides induced by NPC-KT appeared to be identical to those induced by P3HR-1 virus. The ability of NPC-KT virus to induce EA was enhanced more than 10-fold by treatment of superinfected cells with dimethyl sulfoxide; however, dimethyl sulfoxide treatment did not enhance superinfection by P3HR-1 virus. After infection, DNA synthesis of both the superinfecting NPC-KT virus and the resident Raji viral genome was induced. In addition to amplified Raji EBV episomal DNA, a fused terminal fragment of NPC-KT viral DNA was detected. The detection of fused terminal DNA fragments suggests that the superinfecting virion DNA either circularizes or polymerizes after superinfection and is possibly amplified through circular or concatenated replicative intermediates.     

Levels of cellular DNA polymerases in synchronized bovine paravovirus-infected cells.

Infection of synchronized bovine fetal spleen cells with bovine parvovirus results in changes in the levels and patterns of DNA polymerases alpha and gamma during the cell cycle. The pattern of DNA polymerase alpha activity closely paralled viral DNA synthesis and the production of progeny virus, and levels, of this enzyme were threefold greater than in mock-infected cells during the period of maximal viral DNA synthesis. DNA polymerase gamma activity remained slightly elevated during viral DNA replication. Levels and patterns of DNA polymerase beta were similar in mock- and virus-infected cells.     

Mechanisms of infection with Epstein-Barr virus. I. Viral DNA replication and formation of noninfectious virus particles in superinfected Raji cells.

Human lymphoblastoid Raji cells, which do not produce virus, supported replication of Epstein-Barr virus (EBV) upon superinfection. Early antigen, viral capsid antigen, and virions were produced in Raji cells superinfected with EBV. Viral DNA replicated under complete inhibition of host cell DNA synthesis to the extent that a few micrograms of EBV DNA were recovered from 107 superinfected Raji cells, corresponding to 5,000 viral genomes/cell. Homology of the synthesized viral DNA to parental EBV DNA was more than 90%. Virions produced by the Raji cells contained a 55S DNA but failed to induce early antigen, viral capsid antigen, and viral DNA synthesis after a second superinfection of Raji cells.     

Nucleosomal structure of Epstein-Barr virus DNA in transformed cell lines.

Micrococcal nuclease digestion was used to analyze Epstein-Barr virus (EBV) DNA structure in nuclei of transformed cells. Digests of virus-producing (P3HR-1), non-virus-producing (Raji), and superinfected Rajii cell nuclei were fractionated by electrophoresis on agarose gels, transferred to nitrocellulose, and hybridized to 32P-labeled EBV DNA. The viral DNA of Raji nuclei produced a series of bands on electrophoresis whose lengths were integral multiples of a unit size, which was the same as the repeat length of host DNA. Viral DNA in nuclei of P3HR-1 and superinfected Raji cells produced faintly visible bands superimposed on a smear of viral DNA which dominated the hybridization pattern. No differences were detected in the patterns when total DNA digests from Raji, P3HR-1, and an EBV DNA-negative cell line (U-698M) were analyzed by ethidium bromide staining or by hybridization with the use of 32P-labeled lymphoblastoid cell DNA as probe. We conclude that the EBV episomal DNA of Raji cells is folded into nucleosomes, whereas most of the viral DNA of P3HR-1 and superinfected Raji cells is not. This pattern of DNA organization differs signficantly from that in papova group viruses.     

Early Events of Fusion Between Epstein Barr Virus and Human Lymphoblastoid Cells (Raji) Detected by R18 Fluorescence Dequenching Measurements

Relief of fluorescence self-quenching was used to monitor fusion (14) of Epstein Barr virus (EBV) with Raji cells after exposure of the virus to a variety of experimental conditions such as neutral or low pH, enzymatic modification of the viral spike glycoproteins, or inhibition of the protein kinase C (PKC) activity. Incubation of the virus at pH 5.9 prior to the binding to the cell membrane led to a significant enhancement of fusion with the plasma membrane. Treatment of Raji cells with an agent known to elevate the endosomal and lysosomal pH (lysosomotropic agent) (3, 12) partially prevented fusion at neutral pH. Desialylation of EBV significantly reduced the extent of fusion with Raji cells. Protein kinase C inhibitor reduced EBV fusion with Raji cells, while treatment with the tumor promoter and the PKC activator TPA caused an increase in the final extent of fusion. Our results suggest that EBV fuses with lymphoblastoid cells in the endocytic vescicles after being rapidly internalized and that protein kinase C is involved in the process of viral entry into cells.     

Early events of fusion between Epstein Barr virus and human lymphoblastoid cells (Raji) detected by R18 fluorescence dequenching measurements

Relief of fluorescence self-quenching was used to monitor fusion (14) of Epstein Barr virus (EBV) with Raji cells after exposure of the virus to a variety of experimental conditions such as neutral or low pH, enzymatic modification of the viral spike glycoproteins, or inhibition of the protein kinase C (PKC) activity. Incubation of the virus at pH 5.9 prior to the binding to the cell membrane led to a significant enhancement of fusion with the plasma membrane. Treatment of Raji cells with an agent known to elevate the endosomal and lysosomal pH (lysosomotropic agent) (3, 12) partially prevented fusion at neutral pH. Desialylation of EBV significantly reduced the extent of fusion with Raji cells. Protein kinase C inhibitor reduced EBV fusion with Raji cells, while treatment with the tumor promotor and the PKC activator TPA caused an increase in the final extent of fusion. Our results suggest that EBV fuses with lymphoblastoid cells in the endocytic vesicles after being rapidly internalized and that protein kinase C is involved in the process of viral entry into cells.     

Characterization of Epstein–Barr virus reactivation in a modeled spaceflight system

Epstein–Barr virus (EBV) is the causative agent of mononucleosis and is also associated with several malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma, among others. EBV reactivates during spaceflight, with EBV shedding in saliva increasing to levels ten times those observed pre‐and post‐flight. Although stress has been shown to increase reactivation of EBV, other factors such as radiation and microgravity have been hypothesized to contribute to reactivation in space. We used a modeled spaceflight environment to evaluate the influence of radiation and microgravity on EBV reactivation. BJAB (EBV‐negative) and Raji (EBV‐positive) cell lines were assessed for viability/apoptosis, viral antigen and reactive oxygen species expression, and DNA damage and repair. EBV‐infected cells did not experience decreased viability and increased apoptosis due to modeled spaceflight, whereas an EBV‐negative cell line did, suggesting that EBV infection provided protection against apoptosis and cell death. Radiation was the major contributor to EBV ZEBRA upregulation. Combining modeled microgravity and radiation increased DNA damage and reactive oxygen species while modeled microgravity alone decreased DNA repair in Raji cells. Additionally, EBV‐infected cells had increased DNA damage compared to EBV‐negative cells. Since EBV‐infected cells do not undergo apoptosis as readily as uninfected cells, it is possible that virus‐infected cells in EBV seropositive individuals may have an increased risk to accumulate DNA damage during spaceflight. More studies are warranted to investigate this possibility. J. Cell. Biochem. 114: 616–624, 2013. © 2012 Wiley Periodicals, Inc.     

Evidence for integrated EBV genomes in Raji cellular DNA.

Human lymphoid cell lines cannot be grown in long-term tissue culture, as a rule, unless the cells have been transformed by Epstein-Barr virus (EBV). The latent EBV DNA in established cell lines, is mainly present as free covalently closed circles but viral DNA sequences with properties of integrated DNA also seem to be present. We have extended the studies on the physical state of the EB viral DNA sequences in the cell line Raji which appear at a lower density than that for free EB viral DNA during fractionation on CsCl density gradients. In such material a novel EcoRI EBV DNA fragment is present, which hybridizes to viral sequences homologous to EcoRI A. This fragment is not present in free covalently closed circular EBV DNA. When this EcoRI fragment is further analysed with HindIII a smaller fragment than expected, which contains BamHI W sequences, is detected. The demonstration of this HindIII fragment and its characteristics as a joint, viral-host chromosome fragment will be discussed.     

Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases.

We have isolated cDNA clones encoding the human DNA polymerase alpha catalytic polypeptide. Studies of the human DNA polymerase alpha steady-state mRNA levels in quiescent cells stimulated to proliferate, or normal cells compared to transformed cells, demonstrate that the polymerase alpha mRNA, like its enzymatic activity and de novo protein synthesis, positively correlates with cell proliferation and transformation. Analysis of the deduced 1462-amino-acid sequence reveals six regions of striking similarity to yeast DNA polymerase I and DNA polymerases of bacteriophages T4 and phi 29, herpes family viruses, vaccinia virus and adenovirus. Three of these conserved regions appear to comprise the functional active site required for deoxynucleotide interaction. Two putative DNA interacting domains are also identified.     

Association of Epstein-Barr virus early antigen diffuse component and virus-specified DNA polymerase activity.

The role of Epstein-Barr virus (EBV) early antigen diffuse component (EA-D) and its relationship with EBV DNA polymerase in EBV genome-carrying cells are unclear, EBV-specified DNA polymerase was purified in a sequential manner from Raji cells treated with phorbol-12,13-dibutyrate and n-butyrate by phosphocellulose, DEAE-cellulose, double-stranded DNA-cellulose, and blue Sepharose column chromatography. Four polypeptides with molecular masses of 110,000, 100,000, 55,000, and 49,000 daltons were found to be associated with EBV-specified DNA polymerase activity. A monoclonal antibody which could neutralize the EBV DNA polymerase activity was prepared and found to recognize 55,000- and 49,000-dalton polypeptides. An EA-D monoclonal antibody, R3 (G. R. Pearson, V. Vorman, B. Chase, T. Sculley, M. Hummel, and E. Kieff, J. Virol. 47:183-201, 1983), was also able to recognize these same two polypeptides associated with EBV DNA polymerase activity. It was concluded that EBV EA-D polypeptides, as identified by R3 monoclonal antibody, are critical components of EBV DNA polymerase.     

Activation of latent Epstein-Barr virus genomes: selective stimulation of synthesis of chromosomal proteins by a tumor promoter.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a potent inducer of Epstein-Barr virus (EBV) gene expression. The optimal conditions for maximum activation of latent EBV genomes by TPA were determined. Although TPA is able to induce replication of EBV genomes in P3HR-1 cells in all phases of growth, the greatest increase in viral genome copies per cell (15-fold above the control level) occurred in nonproliferating cells as opposed to cells growing exponentially (6-fold above the control level). The synthesis of chromosomal proteins in nonproliferating cells under the conditions that induce maximum activation of latent virus genomes by TPA was studied. Selective stimulation in chromosomal protein synthesis accompanied the increase in EBV genomes in P3HR-1 cells despite an overall reduction in total cellular protein synthesis. Comparison of the chromosomal proteins from TPA-induced P3HR-1 cells and from superinfected Raji cells revealed comigrating chromosomal polypeptides of 145K, 140K, 135K, 110K, 85K, and 55K that are presumably EBV associated. The selective stimulation of synthesis of these chromosomal proteins in TPA-treated P3HR-1 cells was closely associated with the activation of latent EBV genomes.     

Separation of Epstein-Barr Virus DNA from Large Chromosomal DNA in Non-virus-producing Cells

AT least four established human lymphocyte cell lines, one that originates from a Burkitt's lymphoma and the others from normal persons, contain Epstein-Barr virus (EBV) genome¹. These cells show no viral antigens by immunofluorescence tests nor do they produce virus particles. We are examining one of the four cell lines, Raji (cells from a Burkitt's lymphoma), in more detail. The DNA isolated from purified Raji chromosomes contains as much virus genome as the DNA extracted from whole cells (65 genome equivalents per cell)¹. The viral DNA therefore seems to be in the chromosomes. This result, however, does not necessarily indicate that the viral DNA is physically integrated into chromosomal DNA. The following experiments suggest that the EBV DNA in Raji cells is not covalently linked to the large chromosomal DNA, although the number of viral genomes per cell remains constant during passage. The results do not, however, exclude the possibility that small fragments of cell DNA are bonded to the viral DNA. The data also indicate that EBV DNA in Raji cells exists in strands of complete or nearly complete size.