Effect of 12-O-tetradecanoyl-phorbol-13-acetate on the replication of Epstein-Barr virus. I. Characterization of viral DNA.

The tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), induces replication of Epstein-Barr virus (EBV) DNA in a virus-producing human lymphoblastoid cell line, P3HR-1, but not in a nonproducer cell line, Raji. A 6-fold increase in EBV genome copies per P3HR-1 cell parallels the increase in percentage of cells synthesizing viral capsid antigen. In situ cytohybridization with EBV-specific cRNA shows that most of the TPA-treated population participates in the virus-productive cycle. In Raji cells there is abortive induction with an increase in cells showing early antigen from <0.01% to approximately 10%, but there is no increase in EBV genome copies per cell. The optimal TPA concentration for induction of viral DNA replication is 10 ng/ml. EBV DNA synthesized in Raji cells superinfected by virus prepared from TPA-induced P3HR-1 cells is increased approximately 15-fold above that of Raji cells superinfected with control virus. The buoyant density of EBV DNA isolated from virus from TPA-induced cells or of DNA from Raji cells superinfected with TPA-induced and control virus is identical; viral DNA from all sources had the same S value. The XhoI restriction endonuclease digestion patterns of TPA-induced viral DNA and control viral DNA were the same as the viral DNA recovered from Raji cells superinfected with TPA-induced and control virus. Some differences were noted in the molar ratios of some of the fragments.     

Epstein-Barr virus DNA synthesized in superinfected Raji cells.

Raji and P3HR-1 are established Burkitt lymphoma-derived cell lines that carry the Epstein-Barr virus (EBV) genome. Superinfection of the Raji cell line, a non-virus-producer, with virus from P3HR-1 cells results in the synthesis of several thousand copies of EBV-DNA per cell with attendant inhibition of synthesis and breakdown of Raji cell DNA. The DNA synthesized in superinfected Raji cells has been characterized. Raji cells infected with P3HR-1 virus and labeled with 32P 10 hr after infection synthesized only viral DNA. As much as 90% of the 55 S, 32P-labeled material was localized in the nucleus of superinfected cells after a 10-hr labeling period. The viral DNA purified from superinfected cells had the same buoyant density as the DNA isolated from P3HR-1 virions and after purification could be recovered with a specific activity exceeding 106 cpm/μg in an amount which approached 10 wg/107 infected Raji cells. The viral DNA from superinfected cells reassociated with the DNA from Raji or P3HR-1 cells and with the DNA from virus but did not reassociate with DNA from cell line 698 (a lymphoblastoid cell line lacking the EBV genome). Nuclei isolated from superinfected Raji cells incorporated label from deoxythymidine triphosphate into an acid-insoluble product, most of which had a buoyant density identical to that of DNA from P3HR-1 virions. Digestion of the DNA from superinfected cells with the restriction endonuclease EcoRI produced a number of fragments with molecular weights which ranged from less than 1 million to approximately 30 million when analyzed by electrophoresis on agarose gels. All of the fragments produced by digestion of DNA from virus were present in the digest of DNA from superinfected Raji cells.     

Expression of altered ribonucleotide reductase activity associated with the replication of the Epstein-Barr virus.

The expression of the Epstein-Barr virus (EBV) genome can be regulated in the epithelial/Burkitt hybrid (D98/HR-1) cell line and Raji lymphoblastoid nonproducer cell line by induction with 5-iododeoxyuridine (IUdR) or by superinfection with EBV. Extracts of control and induced D98/HR-1 and Raji cells were assayed for ribonucleotide reductase activity in the presence and absence of hydroxyurea (HU). Enzyme activity of control D98/HR-1 and Raji cells was inhibited by greater than 70%, by HU at both low (2 × 10^?4 M) bot high (5 × 10^?4 M) concentrations; however, the reductase activities of IUdR-induced D98/HR-1 cells, superinfected Raji cells, and IUdR-induced Raji cells were resistant to both levels of HU, and enzyme activities of 85 to +100% of control values were obtained in all cases. Under conditions that allowed only partial expression of the EBV genome (before removal of IUdR; early time after superinfection), very significant levels (70–85°k) of HU-resistant enzyme activity were obtained in the presence of 2 × 10^?4 M HU, whereas only 30 to 45% of control reductase activity was observed at the high HU concentration. Mixing experiments employing combinations of various D98/HR-1 control and induced cell extracts indicated that the HU-resistant reductase activity present in induced 1)98/HR-1 cells was due to the presence of an altered enzyme activity and not due to some nonenzymatic factor(s). Additional experiments, in which HU was preincubated with control and induced cell extracts, showed that the HU-resistant ribonucleotide reductase in extracts of induced D98/HR-1 cells was not due to inactivation of the inhibitor by an enzyme or factor present in these cells. These findings of an altered ribonucleotide reductase activity associated with EBV replication, considered in light of similar findings for equine herpesvirus type 1 and herpes simplex virus types 1 and 2, suggest that alteration of this enzyme activity may be a feature of herpesvirus replication.     

Characterization of DNA from polyhedral inclusion bodies of the nucleopolyhedrosis single-rod virus pathogenic for Orgyia pseudotsugata.

A nucleotide sequence complexity of 88.5 × 10⁶ was determined for the DNA of the nucleopolyhedrosis single-rod (unicapsid) virus of Orgyia pseudotsugata using optical renaturation. In addition, the genome size was determined to be 85 × 10⁶ by comparison of EcoR1 restriction endonuclease fragments with markers of known size using agarose gel electrophoresis. A G + C concentration of 44% for the viral DNA was estimated from its melting properties and buoyant density in CsCl. Evidence from buoyant density in CsCl indicates that DNA which is occluded in the polyhedral matrix but not associated with virions is of viral origin.     

Improved production of Epstein-Barr virus DNA for nucleic acid hybridization studies.

Large quantities of Epstein-Barr virus (EBV) DNA were prepared by superinfection of Raji cells with EBV. Virus DNA thus prepared showed a single symmetrical peak at 1.718 g/cm³ in CsCl analytical centrifugation and served as a good template for preparation of cRNA specific to EBV DNA. The virus DNA was also highly labeled in vivo with [³H]thymidine or ³²PO₄ for DNA-DNA reassociation studies. The yield of viral DNA from Raji cells, superinfected with EBV prepared from supernatant fluid of HRI cell culture, was 100- to 570-fold higher than that of viral DNA directly extracted from the same virus preparation.     

Monoclonal antibody specific for capsid antigen of Epstein-Barr virus

A hybrid cell line (Cl-5l) producing an anti-capsid antibody was obtained by fusion of mouse myeloma cells with spleen cells from mice immunized with purified P3HR-1 Epstein-Barr virus (EBV). Immunofluorescence showed that the Cl-5l antibody reacted with the cytoplasm and the nucleus of P3HR-1 and B95-8 cells, but not with Raji, BJAB, Molt-4, and superinfected Raji cells in the presence of cytosine arabinoside (Ara-C). The viable P3HR-1 and B95-8 cells were not stained nor was the viral infectivity neutralized. The Cl-5l antibody immunoprecipitated 123,000 and 120,000 dalton polypeptides of P3HR-1 and B95-8 cells, respectively, and both were sensitive to phosphonoacetic acid. Specific reactions were not evident with extracts of Raji cells and superinfected Raji cells in the presence of Ara-C. An analysis of the purified virus particles showed that this antibody recognized a capsid component of EBV.     

Antigenic variation in alkaline deoxyribonuclease induced by three different strains of Epstein-Barr virus

To determine whether biological and/or biochemical variants exist between strains of Epstein-Barr virus (EBV), we superinfected Raji cells with the nontransforming lytic strain of EBV (HR-1), and two isolates that both transform B-lymphocytes and superinfect Raji cells, B95–8, and NPC-EBV. The superinfected cells were assayed for EBV specific DNase. A new electrophoretic form of DNase was observed in cells superinfected with B95-8 EBV as compared to the enzymes induced by the HR-1 and NPC-EBV isolates. There were antigenic differences in the DNase induced by the EBV strains. Since antibody to EBV DNase is a marker for nasopharyngeal carcinoma (NPC), these data may have implications for EBV-associated disease.     

Physical characterization of a stomatitis papulosa virus genome: A cleavage map for the restriction endonucleasesHindIII andEcoRI

Summary The genome of stomatitis papulosa virus (aparapoxvirus) was cleaved with the restriction endonucleasesHindIII andEcoRI, each giving rise to 6 fragments respectively. Double digestion with both enzymes resulted in 8 bands, two of which contained DNA fragments in double molar concentrations as revealed by reciprocal digests of isolated DNA fragments. The genome size, estimated by summation of the molecular weights of the fragments, is approximately 86×10⁶ daltons, some 30×10⁶ daltons smaller than vaccinia virus (anorthopoxvirus) DNA.The cleavage sites ofHindIII andEcoRI endonucleases were mapped on the genome by analysis of reciprocal digests of isolated DNA fragments and by cross-hybridization experiments. This yielded two mapped segments which were then oriented relative to one another by cleavage of isolated partial digestion products.The terminal restriction fragments show rapid renaturation after alkali denaturation and subsequent neutralization, indicating that stomatitis papulosa virus DNA contains terminal cross-links analogous to those found in vaccinia virus DNA.With 9 Figures     

Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by n-butyrate.

n-Butyrate was found to increase the number of virus producer cells to a dramatic extent in the Epstein-Barr virus (EBV)-carrying P3HR-1 and B95-8 lines. Induction was also seen in the nonproducer Raji and the low producer Daudi lines, but at a mucch lower level. The virus containing supernatant of the butyrate treated P3HR-1 cells induced preferentially EBNA in EBV-negative Ramos target cells, whereas the spontaneously produced virus induced predominantly EA in Raji indicator cells. This suggests a possible difference in the biological properties of the butyrate induced vs the prototype virus. In addition to providing a convenient method to obtain a high yield of viral-DNA and virus antigen-producing cells in the severely restricted EBV system, the findings raise interesting questions on the mechanism of EBV induction, and its possible relationship to the known differentiation inducing ability of n-butyrate.     

DNA from two Orgyia pseudotsugata baculoviruses: molecular weight determination by means of electron microscopy and restriction endonuclease analysis.

Both aqueous and urea-formamide procedures for spreading nucleic acid were employed for electron microscopic studies on the DNAs from the nucleopolyhedrosis bundle virus (NPBV) and the nucleopolyhedrosis single-rod virus (NPSV) both of which are pathogenic for Orgyia pseudotsugata. The molecular weight estimates via electron microscopy were derived by comparison of the mean length values for the double-stranded relaxed circular DNAs with that of SV40 DNA. The aqueous and urea-formamide spreading methods yielded NPSV DNA molecular weight values of 103 × 10⁶ and 104 × 10⁶ daltons, respectively, and molecular weight values of 87 × 10⁶ and 85 × 10⁶ daltons for NPBV DNA. These molecular weights were compared with molecular weight estimates from restriction endonuclease analysis, sedimentation analysis, and renaturation kinetic analysis. DNA located within the NPBV polyhedra but external to virions was characterized by restriction endonuclease analysis and examined by electron microscopy. It was determined to be composed of fragments of random size and to be viral origin.