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.     

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.     

Epstein-Barr virus-specific DNase activity in nonproducer Raji cells after treatment with 12-o-tetradecanoylphorbol-13-acetate and sodium butyrate.

An Epstein-Barr virus (EBV)-specific DNase was induced in EBV nonproducer Raji cells after treatment with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate. The increase in EBV DNase activity was related to the appearance of early antigen-positive cells. The enzyme had a sedimentation coefficient of 4S and was resistant to 300 mM KCl, and its induction did not depend on viral DNA synthesis. The EBV-specific DNase activity was specifically inhibited by sera from patients who had nasopharyngeal carcinoma with high early antigen activities but not by sera from normal, healthy individuals. There was a correlation between the degree of anti-EBV DNase activity and the titers of early antigen antibody.     

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 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.     

Epstein-Barr virus DNA. IX. Variation among viral DNAs from producer and nonproducer infected cells.

A comparative analysis of three Epstein-Barr virus DNAs from American patients with infectious mononucleosis (B95-8, Cherry, and Lamont) and four Epstein-Barr virus DNAs from African patients with Burkitt lymphoma (AG876, W91, Raji, and P3HR-1) indicated that the usual format of Epstein-Barr virus DNA includes a variable number of direct repeats of a 0.35 X 10(6)-dalton sequence (TR) at both ends of the DNA, a 9 X 10(6)-dalton sequence of largely unique DNA (Us), a variable number of repeats of a 2 X 10(6)-dalton sequence (IR), and a 89 X 10(6)-dalton sequence of largely unique DNA (UL). Within UL there was homology between DNA at 26 X 10(6) to 28 X 10(6) daltons and DNA at 93 X 10(6) to 95 X 10(6) daltons. The relative sequence order (TR, US, IR, UL, TR) did not vary among "standard" Epstein-Barr virus DNA molecules of each isolate. B95-8 DNA had an unusual deletion extending from 91 X 10(6) to 100 X 10(6) daltons, and P3HR-1 DNA had an unusual deletion extending from 23.5 X 10(6) to 26 X 10(6) daltons. There was sufficient variability among the EcoRI and BamHI fragments of the DNAs to identify each isolate specifically. However, we discerned no distinguishing features for the two geographic or pathogenic origins of the seven isolates. Three intracellular DNAs (Raji, Lamont, and Cherry) and one virion DNA (P3HR-1) were heterogenous in molecular organization and had subpopulations of rearranged or defective molecules. Some regions, particularly 59 X 10(6) to 63 X 10(6) daltons and sequences around TR, frequently participated in rearrangements. Restriction endonuclease maps of the standard and rearranged DNAs of the seven isolates are presented.     

The lytic cycle of Epstein-Barr virus in the nonproducer Raji line can be rescued by the expression of a 135-kilodalton protein encoded by the BALF2 open reading frame.

In Epstein-Barr virus (EBV)-carrying nonproducer Raji cells, the induction of the viral replicative cycle by chemical treatment is limited to only the early stage and viral DNA synthesis is totally inhibited. We previously showed the absence of two messenger RNAs that are encoded by the BamHI-A fragment of the EBV genome and that correspond to open reading frames BALF2 and BARF1 in chemically induced Raji cells. Since the BALF2 gene encodes a 135-kDa DNA-binding protein which was immunoprecipitated by antibody against ICP8 protein, a key protein in herpes simplex virus replication, we asked whether the lack of productive cycle in Raji cells is due to the absence of expression of the BALF2 gene. We transfected the Raji cell line with the BALF2 gene. After chemical induction, the BALF2-transfected cells expressed not only early antigens but also late antigens. In these cultures, the viral particles were detected by electron microscopy. The expression of late antigens was completely inhibited by arabinofuranosylthymine, which is a specific inhibitor of viral DNA replication. The BALF2 gene might play an essential role in the induction of the EBV-lytic cycle.     

Different activation of Epstein-Barr virus immediate-early and early genes in Burkitt lymphoma cells and lymphoblastoid cell lines.

Specific expression of the Epstein-Barr virus (EBV) immediate-early and early gene products Zta, Rta, I'ta, and MSta by a recombinant vaccinia virus system allowed us to analyze the first steps in the induction of the lytic cycle in EBV-infected Burkitt lymphoma (BL) cells and lymphoblastoid cell lines (LCLs). Significant differences in the induction of early genes were found between these cell types: whereas in BL cells the trans activator Zta was found to induce key steps of the early lytic cycle, only minor activities of Zta were noted in LCLs. Contrary to Zta, the trans activator Rta was found to be highly effective in LCLs. These observations suggest that Rta may play an important role in the activation of the early lytic cycle in LCLs, although it cannot be activated by Zta. The latter may be a reason for the lower tendency of LCLs to switch into the lytic cycle compared with BL cells or differentiated epithelial 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.     

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.     

Mutant carrying deletions in the two simian virus 40 early genes.

We isolated a simian virus 40 mutant, dl2194, which carried deletions in both early genes. One deletion removed 234 base pairs in the 54/59 region within the small-t-antigen coding sequence and the large-T-antigen gene intron. The second deletion removed 57 base pairs at the C terminus of the large-T-antigen coding sequence (0.20 map unit). dl2194 was a viable mutant, it carried a normal helper function for adenovirus growth on monkey cells, and it displayed the transformation properties of a small-t-antigen-negative single mutant. Therefore, none of the known large-T-antigen functions seemed to be altered by the C-terminal deletion.     

Presence of free viral DNA in simian virus 40-transformed nonproducer cells.

Extracts from several simian virus 40 (SV40)-transformed nonproducer cells were prepared by the hot-phenol procedure normally used to extract cellular RNA. These extracts contained SV40 infectious units. Part of the infectious units were identified as SV40 form I DNA molecules. The results of reconstruction experiments suggest that SV40 form I DNA is extractable by the hot-phenol procedure because of its fast renaturation rate. The significance of the presence of free viral DNA in nonproducer transformed cells is discussed.     

Cell cycle-dependent expression of early viral genes in one group of simian virus 40-transformed rat cells.

SV40-transformed FR 3T3 rat cells were previously shown to exhibit different patterns of accumulation of the virus-coded T-antigen. One group of transformants accumulates T-antigen throughout the cell cycle, whereas in another group, only the cells in the G2 phase of the cell cycle are stained by immunofluorescence with anti-T antigen antibodies. We investigated the mechanism involved by determining the amounts of early SV40 RNA during the cell cycle. Cells in the various phases of the cell cycle were sorted from an asynchronously growing population using a flow cytofluorimeter. Determination of the amounts of viral RNA in the different nuclear and cytoplasmic RNA fractions showed that in transformants with a G2-restricted accumulation of T-antigen, viral RNA was present in G2, to some extent in S, but could not be detected in cells in G1. In contrast, equivalent amounts of viral RNA were detected in all the phases of the cell cycle in the other group of transformants. Cell sorting, performed after pulse-labeling the cells for 2 h with [35S]methionine, confirmed that translation of the viral mRNAs occurred only in G2 in the first group of transformants, and throughout the cell cycle in the second group.     

Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.     

Identification of proteins encoded by Epstein-Barr virus trans-activator genes.

Specific antisera were generated to characterize Epstein-Barr virus proteins reported to have trans-activating properties. Open reading frame BRLF1 was found to be expressed in two modifications in vivo, with molecular sizes ranging from 94 to 98 kilodaltons (kDa) depending on the cell line, whereas only one protein (Raji cells, 96 kDa) was detected by in vitro translation. Open reading frame BZLF1 encoded polypeptides of 38 and 35 kDa and additional smaller forms. A BZLF1-encoded 30-kDa protein could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be shown for the proteins derived from reading frames BZLF1 and BMLF1. BMLF1 expression gave a heterogeneous protein pattern, with molecular sizes between 45 and 70 kDa, including a predominant 60-kDa protein detected in different B-cell lines.     

Epstein-Barr virus and the somatic hypermutation of immunoglobulin genes in Burkitt's lymphoma cells

It has been suggested that Epstein-Barr virus (EBV) might suppress antibody maturation either by facilitating bypass of the germinal center reaction or by inhibiting hypermutation directly. However, by infecting the Burkitt's lymphoma (BL) cell line Ramos, which hypermutates constitutively and can be considered a transformed analogue of a germinal center B cell, with EBV as well as by transfecting it with selected EBV latency genes, we demonstrate that expression of EBV gene products does not lead to an inhibition of hypermutation. Moreover, we have identified two natural EBV-positive BL cell lines (ELI-BL and BL16) that hypermutate constitutively. Thus, contrary to expectations, EBV gene products do not appear to affect somatic hypermutation.     

Human B cells on their route to latent infection--early but transient expression of lytic genes of Epstein-Barr virus

Epstein-Barr virus (EBV) is a human tumor virus and a paradigm of herpesviral latency. Mature naïve or memory B cells are EBV's preferred targets in vitro and in vivo. Upon infection of any B cell with EBV, the virus induces cellular proliferation to yield lymphoblastoid cell lines (LCLs) in vitro and establishes a latent infection in them. In these cells a ‘classical’ subset of latent viral genes is expressed that orchestrate and regulate cellular activation and proliferation, prevent apoptosis, and maintain viral latency. Surprisingly, little is known about the early events in primary human B cells infected with EBV. Recent analyses have revealed the initial but transient expression of additional viral genes that do not belong to the ‘classical’ latent subset. Some of these viral genes have been known to initiate the lytic, productive phase of EBV but virus synthesis does not take place early after infection. The early but transient expression of certain viral lytic genes is essential for or contributes to the initial survival and cell cycle entry of resting B cells to foster their proliferation and sustain a latent infection. This review summarizes the recent findings and discusses the presumed function(s) of viral genes expressed shortly but transiently after infection of B-lymphocytes with EBV.