Epstein-Barr virus RNA. VI. Viral RNA in restringently and abortively infected Raji cells.

Nuclear and polyadenylated RNA fractions of Raji cells are encoded by larger fractions of Epstein-Barr virus DNA (35 and 18%, respectively) than encode polyribosomal RNA (10%). Polyribosomal RNA is encoded by DNA mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons. An abundant, small (160-base), non-polyadenylated RNA encoded by EcoRI fragment J (0.05 X 10(8) to 0.07 X 10(8) daltons) is also present in the cytoplasm of Raji cells. After induction of early antigen in Raji cells, there was a substantial increase in the complexity of viral polyadenylated and polyribosomal RNAs. Thus, nuclear RNA was encoded by 40% of Epstein-Barr virus DNA, and polyadenylated and polyribosomal RNAs were encoded by at least 30% of Epstein-Barr virus DNA. Polyribosomal RNA from induced Raji cells was encoded by Epstein-Barr virus DNAs mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons and also by DNAs mapping within the long unique regions of Epstein-Barr virus DNA at 0.39 X 10(8) to 0.49 X 10(8), 0.51 X 10(8) to 0.59 X 10(8), 0.66 X 10(8) to 0.77 X 10(8), and 1.02 X 10(8) to 1.05 X 10(8) daltons.     

Cocaine potentiates the switch between latency and replication of Epstein-Barr virus in Raji cells.

This paper shows that cocaine amplifies Epstein-Barr virus (EBV) reactivation in Raji cells. Its effect on early viral protein synthesis was maximal when it was added with 12-O-tetradecanoyl phorbol-13-acetate (TPA) plus n-butyrate, but nil when added alone. The enhancing effect of cocaine on early replicative stages of latent EBV was associated with an increase of Ca(2+) mobilization induced by the drug and with an induction of cellular protein phosphorylation in chemicals and cocaine-treated Raji cells. Cocaine also acted synergistically with TPA and n-butyrate to induce Z Epstein-Barr replication activator (ZEBRA), a nuclear phosphoprotein responsible for the activation of early viral gene expression. These findings provide the first evidence that cocaine may represent an important co-factor in the reactivation of early stages of latent EBV infection.     

Epstein-Barr virus-specific DNA polymerase in virus-nonproducer Raji cells.

Virus-nonproducer Raji cells, when induced to early antigen synthesis by 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate, showed an increase in DNA polymerase activity. This enzyme has the characteristics of a typical Epstein-Barr virus DNA polymerase with regard to chromatographical pattern and biological properties: it is eluted from DEAE-cellulose at 0.08 M NaCl, has a high salt resistance, is sensitive to phosphonoacetic acid and phosphonoformate, and shows a substrate preference for poly(dC)-oligo(dG12-18). The resistance of Epstein-Barr virus polymerase activity to aphidicolin is a property distinct from that of HSV DNA polymerase. Viral DNA polymerase activity increases in the absence of Epstein-Barr virus DNA replication, indicating that this enzyme is an early viral protein.     

Replication of latent Epstein-Barr virus genomes in Raji cells.

The replication of the 50 to 60 latent, predominantly extrachromosomal, Epstein-Barr virus genomes maintained by the Burkitt-lymphoma-derived Raji cell line was investigated by using a Meselson-Stahl density transfer approach. Samples of DNA isolated from cells cultivated for different periods in bromodeoxyuridine-supplemented medium were fractionated according to density, and the distribution of viral and cellular DNAs among the heavy-, hybrid-, and light-density species was quantitated. The results indicate that the majority of latent Epstein-Barr virus DNA plasmids each replicate once during the cell cycle.     

Sedimentation characteristics of newly synthesized Epstein-Barr viral DNA in superinfected cells.

Replicating Epstein-Barr virus (EBV) DNA molecules isolated from superinfected Raji cells were shown to consist of 80S to 65S and 58S (mature) molecules Pulse-chase experiments showed that radioactive label of DNAS molecules with the larger sedimentation coefficients was partially chased into 58S labeled forms. Formation of large concatemers of viral DNA could not be detected at any time after superinfection. The continuous presence of the 65S viral DNA intermediate throughout the replicative cycle combined with the observed inhibition of EBV DNA synthesis by addition of nontoxic levels of ethidium bromide to the superinfected cell culture led us to propose that EBV replication proceeds via a relaxed circular DNA intermediate.     

Effect of the arginine analog canavanine on the synthesis of Epstein-Barr virus-induced proteins in superinfected Raji cells.

The addition of canavanine to cultures of superinfected Raji cells in the absence of arginine prevented the appearance of early antigens as defined by immunofluorescent staining. Addition of various amounts of arginine permitted the identification of at least three groups of proteins, each responding differently to the various concentrations of arginine-canavanine.     

C-1027-induced alterations in Epstein-Barr viral DNA replication in latently infected cultured human Raji cells: relationship to DNA damage.

This study is the first detailing drug-induced changes in EBV DNA replication intermediates (RIs). Both EBV replication inhibition and damage induction were studied in latently infected human Raji cells treated with the enediyne DNA strand-scission agent C-1027. Analysis of RIs on two-dimensional agarose gels revealed a rapid loss in the EBV bubble arc. When elongation of nascent chains was blocked by aphidicolin, this loss was inhibited, suggesting that C-1027-induced disappearance of RIs was related to maturation of preformed replication molecules in the absence of initiation of new RIs. C-1027 damage to EBV DNA was limited at concentrations where loss of the bubble arc was nearly complete, and none was detected within the replicating origin (ori P)-containing fragment, indicating that replication inhibition occurred in trans. By contrast, the non-nuclear mitochondrial genome was insensitive to replication inhibition but highly sensitive to damage induced by C-1027. C-1027-induced trans inhibition of nuclear but not mitochondrial DNA replication is consistent with a cell cycle checkpoint response to a DNA-damaging agent. EBV replication and Raji cell growth were inhibited at equivalent C-1027 doses.     

Epstein-Barr virus gene expression in P3HR1-superinfected Raji cells.

The pattern of Epstein-Barr virus (EBV) RNAs expressed in Raji cells superinfected with P3HR1 EBV was examined. RNAs whose expression was of an immediate-early type (resistant to treatment of the cells with anisomycin) were identified. These RNAs, encoding the EBV reading frames BZLF1 and BRLF1, were probably expressed from defective virus within the P3HR1 preparation, and some of them were responsible for the induction of the EBV productive cycle in the Raji cells. The structures of the B95-8 RNAs equivalent to the anisomycin-resistant RNAs were determined. The RNA encoding the BZLF1 reading frame contained two splices which extended and modified the reading frame from that previously described.     

Plasticity of DNA replication initiation in Epstein-Barr virus episomes

In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD) to study the latent duplication of Epstein-Barr virus (EBV) episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size). These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes.     

Studies of the Epstein-Barr virus receptor found on Raji cells. I. Extraction of receptor and preparation of anti-receptor antibody

Raji, a human lymphoblastoid cell line, expresses a membrane receptor (EBVR) specific for Epstein Barr virus (EBV). A component that binds EBV was extracted from this cell line by treatment of the cells for 3 hr on ice with Tris buffer containing 10% glycerol. The treatment reduced the capacity of the cells to bind virus, and after concentration the receptor extract (RE) inhibited both EBV binding and superinfection of fresh Raji cells. Similarly prepared extracts of EBVR- cells lacked such activity. An antibody was made to the extract (anti-RE), which after absorption with EBVR- cells, bound to the same percentages of EBVR+ lymphoblastoid cell lines, EBVR+ human/mouse somatic cell hybrids, and fresh peripheral B cells as the virus did. In reciprocal assays, preincubation of EBVR+ cells with anti-RE inhibited virus binding. Doubly stained patches were observed on membranes of EBVR+ cells that had been incubated simultaneously with virus and anti-RE and stained respectively with rhodaminated and fluoresceinated reagents. The major polypeptide immunoprecipitated by anti-RE from radiolabeled Raji cells had an approximate calculated m.w. of 150,000.     

Sequence requirements of the Epstein-Barr virus latent origin of DNA replication.

The Epstein-Barr virus (EBV) latent origin of DNA replication (oriP) is composed of two elements that contain binding sites for the sole viral gene product required for latent cycle replication, EBNA-1. One of these elements, region I, functions as an EBNA-1-dependent enhancer for RNA polymerase II-transcribed genes, may play a role in plasmid segregation, and is required for origin function in B cells latently infected with EBV. The second element, region II, contains or is very near the site of initiation of DNA replication. A genetic approach was taken to determine the contribution of the EBNA-1 binding sites in oriP to origin function. Although region I is required for the transient replication of plasmids bearing region II in EBV-infected B cells, a plasmid lacking region I but containing region II, was observed to replicate transiently in both D98/Raji and HeLa cells expressing EBNA-1. Thus, binding of EBNA-1 to region I is not absolutely required for the molecular events that lead to initiation of DNA replication at region II. Site-directed mutagenesis of the four EBNA-1-binding sites in region II, individually and in various combinations, demonstrated that only two EBNA-1-binding sites are required for region II function. The results obtained with these mutants, together with the analysis of the replicative ability of plasmids containing insertions between EBNA-1-binding sites, suggested that the spatial relationship of the two sites is critical. Mutants that contain only two EBNA-1-binding sites separated by 26 to 31 bp in region II were not maintained as plasmids over many cell generations and were greatly reduced in their ability to replicate transiently in D98/Raji cells. The EBNA-1-induced bending or untwisting of the DNA in EBNA-1-binding sites 1 and 4 in region II did not, however, demonstrate this spatial constraint. It may be concluded from these results that specific protein-protein interactions between EBNA-1 and/or between EBNA-1 and a cellular protein(s) are required for origin function.     

Efficient expression of an Epstein-Barr nuclear antigen in Drosophila cells transfected with Epstein-Barr virus DNA.

In a search for exogenous promoters which function in cultured Drosophila cells, we have co-transfected a D. melanogaster cell line with an Epstein-Barr virus (EBV) cosmid clone which encodes the Epstein-Barr nuclear antigen (EBNA-1). Here we report that Drosophila cells containing stably integrated copies of EBNA-1 encoding DNA synthesise a polypeptide of mol. wt. identical to that of authentic EBNA-1, which is detectable with EBNA-positive but not EBNA-negative human serum. As in EBV-transformed lymphoblastoid cells, this neo-antigen is associated with the nucleus of transfected cells suggesting that cellular localisation signals which operate in mammalian cells are also recognised in insect cells.     

Host factors associated with the kinetics of Epstein-Barr virus DNA load in patients with primary Epstein-Barr virus infection

The aims of this study were to elucidate the kinetics of Epstein-Barr virus (EBV) DNA load in serially collected peripheral blood mononuclear cells of patients with primary EBV infection, and to determine the correlated host factors. Blood samples were collected from 24 patients with primary EBV infection. EBV DNA copy numbers were measured using real-time polymerase chain reaction. Based on the kinetics of EBV DNA load, the 24 patients were divided into two groups: rapid regression and slow regression. Eighteen of the 24 patients (75%) were included in the slow regression and 6 (25%) in the rapid regression group. No statistically significant differences were observed between the two groups in clinical features and laboratory findings. However, acute phase (3 to 10 days after the onset of the illness) serum samples from six children in the slow regression and four in the rapid regression group revealed significantly higher serum interleukin (IL)-1β (P= 0.018), IL-12 (P= 0.009), tumor necrosis factor-α (P= 0.019), interferon-inducible protein 10, and monokine induced by interferon γ concentrations in the rapid regression than the slow regression group. On the other hand, sera from six children in the slow regression and four in the rapid regression group in the convalescent phase (14 to 21 days after the onset of the illness) showed no statistically significant differences between the two groups in these biomarker concentrations. Based on this, it was concluded that the kinetics of EBV DNA load can be divided to two different patterns after primary EBV infection, and immune response might be associated with viral clearance.     

Interaction with the Epstein-Barr virus helicase targets Zta to DNA replication compartments

Zta has a dual role in the Epstein-Barr virus (EBV) lytic cycle, acting as a key regulator of EBV lytic gene expression and also being essential for lytic viral DNA replication. Zta's replication function is mediated in part through interactions with the core viral replication proteins. We now show interaction between Zta and the helicase (BBLF4) and map the binding region to within amino acids (aa) 22 to 86 of the Zta activation domain. In immunofluorescence assays, green fluorescent protein (GFP)-tagged BBLF4 localized to the cytoplasm of transfected cells. Cotransfection of Zta resulted in translocation of BBLF4-GFP into the nucleus indicating interaction between these two proteins. However, Zta with a deletion of aa 24 to 86 was unable to mediate nuclear translocation of BBLF4-GFP. Results obtained with Zta variants carrying deletions across the aa 24 to 86 region indicated more than one contact site for BBLF4 within this domain, and this was reinforced by the behavior of the four-point mutant Zta (m22/26,74/75), which was severely impaired for BBLF4 interaction. Binding of BBLF4 to Zta was confirmed using GST affinity assays. In both cotransfection-replication assays and replication assays performed in EBV-positive P3HR1 cells, the Zta (m22/26,74/75) mutant was replication defective. In Zta-transfected D98-HR1 cells, replication compartments could be detected by immunofluorescence staining using anti-BMRF1 monoclonal antibody. Cells transfected with Zta variants that were defective for helicase binding still formed replication compartments, but Zta was excluded from these compartments. These experiments reveal a role for the Zta-helicase interaction in targeting Zta to sites of viral DNA replication.     

Alternate replication in B cells and epithelial cells switches tropism of Epstein-Barr virus

Epstein-Barr virus is ubiquitous and is causally implicated in lymphoid and epithelial malignancies. Virus invades oropharyngeal mucosa and establishes latency in B lymphocytes. Reactivating lymphocytes shed virus into saliva for spread to new hosts. A complex of three virus glycoproteins, gH, gL and gp42, is essential for entry. B-cell entry requires binding of gp42 to human leukocyte antigen (HLA) class II whereas entry into epithelial cells lacking HLA class II requires complexes without gp42. To accommodate infection of each, the virus carries both three-part and two-part complexes. We show here that HLA class II in the virus-producing cell alters the ratio of three-part to two-part complexes. As a consequence, virus originating in epithelial cells efficiently infects B cells whereas B-cell derived virus better infects epithelial cells. This molecular switch is a novel strategy that could alter tropism of virus from epithelium to B cells and then back to epithelium in a new host.     

Primer-DNA formation during simian virus 40 DNA replication in vitro.

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.     

Induction of Epstein-Barr virus nuclear antigen and DNA synthesis in a human epithelial cell line after Epstein-Barr virus infection.

The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma is supported by the presence of EBV genomes in the epithelial elements of the tumor and by elevated antibody titers to EBV-specific antigens in the patients; the levels of these titers are related to the clinical course of the disease. However, since most laboratory data suggest that EBV is a B-lymphotropic virus, it is unclear how the virus becomes associated with the epithelial elements of the nasopharynx. The purpose of the present work was to find a human model system to study this association. A human epithelial line (U) was found that could be directly infected by EBV, and viral functions, the induction of EBV nuclear antigen and cellular DNA synthesis, were demonstrated. The U line was established in 1957 by the late H. J. Van Kooten (Kok-Doorschodt at the University of Utrecht), and although it is no longer diploid, it exhibits density inhibition. When U cells were infected with EBV, EBV nuclear antigen was expressed in 6 to 16% of the cells, 1 and 2 days after infection with B95-8 virus, but not with the P3HR-1 strain. No evidence for virus replication was obtained; immunofluorescence staining for early antigens and virus capsid antigens gave negative results. Quantitative adsorption experiments for EBV indicated that the adsorption capacity of U cells is significant (60% of Raji cells). The present results also demonstrated that infection with the virus overcomes block(s) in cellular DNA synthesis caused by 5-fluorodeoxyuridine. The induction of DNA synthesis was determined by increased incorporation of [3H]thymidine into the cells. The highest level of isotope incorporation was observed at about 15 h after infection and thereafter decreased. Analysis of the induced DNA indicated that it was of cellular origin.