Ronald Glaser

Expression of altered ribonucleotide reductase activity associated with the the replication of the epstein-barr virus

Abstract 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.

Studies of an epstein-barr virus-induced DNA polymerase

Abstract Epithelial/Burkitt hybrid cells (D98/HR-1) typically express the Epstein-Barr-associated nuclear antigen (EBNA). Synthesis of early antigen (EA) and virus capsid antigen (VCA) is repressed. Following treatment with 5-iodo-2′-deoxyuridine (IUdR), however, Epstein-Barr virus (EBV) EA and late virus-specific products, including EBV DNA and complete virions, are induced. This report demonstrates that phosphonoacetic acid inhibits expression of VCA, but has no effect on EA. Cells treated with IUdR were also found to express a new DNA polymerase which was susceptible to stimulation by salt and was not present in uninduced cells. This salt-stimulated DNA polymerase resembles other herpesvirus-induced DNA polymerases in its requirements for maximal activity and its ability to use synthetic templates in DNA synthesis. Data suggest that this DNA polymerase is induced by EBV and is necessary for the productive synthesis of virus-specific DNA.

Epstein-Barr Virus: Detection of Genome in Somatic Cell Hybrids of Burkitt Lymphoblastoid Cells

Somatic cell hybrids of Burkitt lymphoblastoid cells, from which Epstein-Barr virus can be recovered, were examined for the presence of virus DNA by DNA-RNA hybridization. Four clones of hybrid cells, each negative for virus antigens by immunofluorescence, contained virus DNA in varying genomic equivalents. The number of virus genome equivalents increased in the hybrid cells after induction of virus with iododeoxyuridine.

Transcription of latent Epstein-Barr virus genomes in human epithelial/Burkitt hybrid cells

Abstract Transcription of latent Epstein-Barr virus genomes in somatic cell hybrids of Burkitt lymphoblastoid cells has been studied. D98/HR-1, D98/Raji, and Raji cells contained viral RNA transcribed from approximately 25% of virus DNA. After iododeoxyuridine (IUdR) induction 50% of virus DNA was found transcribed in these three cell lines. Raji cells, however, showed production of early antigens (EA) only, whereas the two other cell lines produced both EA and virus capsid antigens, suggesting that there may be some post-transcriptional control in Roji cells after IUdR induction. The amount of virus RNA in the cells calculated from DNA-RNA hybridization kinetics was proportional to the number of virus genomes per cell. This suggests that a constant fraction of the multiple copies of virus DNA is transcribed in the three different cell systems.

Observations on the resistance of Epstein-Barr virus DNA synthesis to hydroxyurea

Abstract The EBV genome was expressed in EBV “negative” D98/HR-1 somatic cell hybrids following treatment with IUdR as monitored by induction of early antigen (EA), virus capsid antigen (VCA), and virus particles. When the hybrid cells were treated with hydroxyurea (HU) following induction, total DNA synthesis was dramatically reduced but expression of both EA and VCA was not altered. The number of EBV genome equivalents increased following induction with IUdR in the presence of 5 mM HU. Synthesis of EBV specific DNA proceeded to the same degree with or without treatment with HU in lymphoblastoid cells infected with EBV while HU treatment reduced total DNA synthesis by 85–90%. Cytosine arabinoside (ara-C) used in the same manner as HU in IUdR induced hybrid cells dramatically reduced cellular DNA synthesis and totally inhibited EBV DNA synthesis. Of all viruses tested thus far, EBV is unique in that EBV specific DNA synthesis is refractile to inhibition by treatment with HU. Several possible explanations for this unique phenomenon are presented.

Thymidine Kinase Activity in Burkitt Lymphoblastoid Somatic Cell Hybrids After Induction of the EB Virus

Summary Epstein-Barr virus (EBV) can be rescued from somatic cell hybrids after treatment with 5-iododeoxyuridine (IUDR). Deoxythymidine kinase activity increased in the hybrid cells after induction of EBV. Activity of deoxycytidine deaminase did not increase significantly following induction of the virus. The evidence suggests that the EBV genome may participate in the increased enzyme activity observed.

Effect of the host cell on the maintenance and replication of Epstein-Barr virus.

Abstract We have studied the role the host cell plays in controlling the expression of the Epstein-Barr virus (EBV) by using Burkitt somatic cell hybrids of human and mouse cells. Mouse/Burkitt somatic-cell hybrids were shown to contain a repressed EBV genome that was inducible with iododeoxyuridine. Electron microscopic examination of human/Burkitt hybrid cells (D98/HR-1 and D98/Raji) and Burkitt lymphoblastoid cells in which EBV was replicating showed an enhancement of virus replication concomitant with an enhancement of EBV-specific cytopathologic effect in D98/HR-1 and D98/Raji cells when compared to Burkitt lymphoblastoid cells. When the stability of the EBV genome in human/Burkitt hybrid cells was studied, it was found that the EBV genome in hybrids of the nonproducer Burkitt cells (Raji) was less stable over time than hybrid cells of producer cells (HR-1). The data obtained in this study support the concept that the cell in which the EBV genome resides plays a major role in the maintenance, expression, and replication of EBV.

Demonstration of a cellular inhibitor of Epstein-Barr and cytomegalovirus synthesis

Abstract A particle free extract prepared from human D98 cells was tested for inhibitory activity against the Epstein-Barr Virus (EBV) and cytomegalovirus (CMV). The number of permissive human cells positive for EBV-specific antigens after infection with virus decreased by more than 90% when the cells were treated with D98 extract and poly- l -ornithine. Synthesis of CMV DNA was depressed in virus infected cells in the presence of the inhibitor. Replication of infectious CMV was reduced by approximately 99.9%. The data suggest that the D98 extract specifically depresses EBV and CMV replication.

Characteristics of the Release of Measles Virus from Latently Infected Cells after Co-Cultivation with BSC-1 Cells

The events during the release of measles virus from latently infected hamster cells after co-cultivation with BSC-1 cells were studied by immunofluorescence and electron microscopy techniques. Before co-cultivation, measles virus antigens (nucleocapsid) were distributed in an apparently random fashion throughout the cytoplasm. Six hours after co-cultivation with BSC-1 cells, the measles virus nucleocapsid became aggregated in close proximity to the nuclear membrane. These antigens then diffused towards the cell periphery, and progeny virus was observed budding from the cell surface by 16 h after co-cultivation. Fusion of the BSC-1 cells to the latently infected hamster cells was necessary for virus release to occur, and an intact, viable BSC-1 cell was also required. A possible mechanism for the block of virus replication in the latently infected cells is discussed.

Oncogenic properties of human viruses

Cancer biology today encompasses a variety of specialties ranging from epidemiology to molecular biology and chemotherapy. One of the very active areas of research is that which supports the concept of a virus etiology for at least some forms of cancer. For several years it has been generally accepted that a variety of both DNA and RNA viruses can transform animal cells in vitro and that they are transplantable and induce tumors in vivo. The virus-transformed cells exhibit changes in growth characteristics typical of cancer cells, and commonly contain virus-specific nucleic acids and antigens. Characterization of tumor viruses and virus-cell associations are being explored by virologists, biochemists, epidemiologists, geneticists, molecular biologists, and immunologists. The nature of the complex problems involved requires the participation of experts in a variety of specialties. In this communication, we present an overview of the status of DNA tumor viruses as they relate to human cancer and to transformation in general. No human C-type RNA virus has yet been identified, although there are reports of primate C-type viruses. The potentially important human papovaviruses, human adenoviruses and the Herpesvirus group, including herpes simplex virus, cytomegalovirus, and the EpsteinBarr virus are discussed. The importance of the Herpesvirus group today as it relates to human neoplasia prompted us to emphasize this area.