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.     

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.     

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.     

Concatameric replication of Epstein-Barr virus: structure of the termini in virus-producer and newly transformed cell lines.

The linear form of Epstein-Barr virus (EBV) DNA has homologous direct tandem repeats of approximately 500 bp at each terminus (TR). After infection, EBV DNA circularizes via the TR to form the intracellular episomal DNA. To analyze the mechanism of the synthesis of linear DNA through possible replicative intermediates, the terminal fragments were identified in the total intracellular DNA and the covalently closed circular DNA from a productively infected cell line after induction of replication or after treatment with an inhibitor of viral DNA synthesis. These studies indicate that some of the fused terminal fragments detected in the total intracellular DNA are replication-dependent forms which are selectively excluded from the covalently closed circular fraction and are eliminated after treatment with acyclovir. The EBV terminal restriction enzyme fragments were identified in three producer cell lines, each with a characteristic number of TR in the intracellular episomal DNA. Identification of the termini in cell lines established with the three virus strains revealed that the newly transformed cell lines had a greater number of TR than did the template DNA in the producer cell line. The increase in the number of TR in progeny episomes indicates that linear DNA is produced from concatameric replicative intermediates rather than from amplified catenated circular intermediates.     

Structure of defective DNA molecules in Epstein-Barr virus preparations from P3HR-1 cells.

Epstein-Barr virus (EBV), isolated from P3HR-1 cells, induces early antigen and viral capsid antigen upon infection of human B-lymphoblasts. The strong early antigen- and viral capsid antigen-inducing activity is only observed in P3HR-1 virus preparations harboring particles with defective genomes, suggesting that this biological activity is directly associated with the defective DNA population. After infection of EBV genome-carrying Raji or EBV genome-negative BJAB cells, defective genomes of P3HR-1 EBV DNA are replicated in excess, depending on the multiplicity of infecting EBV particles. Hybridization of the DNA from such infected cells with 32P-labeled EBV DNA after HindIII cleavage reveals six hypermolar fragments. Mapping of these fragments shows that they form one defective genome unit containing four nonadjacent regions (alpha, beta, gamma, and delta) of the nondefective P3HR-1 EBV DNA. Two of the segments (alpha and beta) contain ca. 17 and 13 megadaltons, respectively, from the terminal regions of the P3HR-1 genome, whereas the two smaller segments (gamma and delta) contain ca. 3.7 and 3.0 megadaltons, respectively, originating from the central portion of the genome. In the defective molecule, the regions gamma and delta are present in the opposite orientation compared with nondefective P3HR-1 EBV DNA. Tandem concatemers are formed by fusion of the alpha and beta regions. Our model suggests that tandem concatemers of three defective genome units can be packaged into virions in P3HR-1 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.     

Localization polymorphism of EBV DNA genomes in the chromosomes of Burkitt lymphoma cell lines

The localization of Epstein-Barr virus (EBV) genomes in nuclei of the human lymphoblastoïd cell lines Raji, Jijoye, P₃HR-1, Daudi and Ramos was investigated by in situ hybridization with biotinylated EBV DNA probes. We found that all sites of hybridization were associated with the chromosomes. Only some of these sites were present on both chromatids and these had a non-random distribution; these sites could represent EBV sequences integrated at specific points on the chromosomes. The total mean site number corresponded with the number of viral DNA copies estimated in the different cell lines by other techniques, but the copy number was highly variable from cell to cell in a given line.     

Epithelial cell retention of transcriptionally active, P3HR-1-derived heterogeneous Epstein-Barr virus DNA with concurrent loss of parental virus

Deleted, rearranged, heterogeneous (het) Epstein-Barr virus (EBV) DNA with the distinctive capability of disrupting EBV latency has been reported in biopsy samples of EBV-associated tumors whose onset in immunocompetent hosts is characteristically preceded by an antibody response indicative of EBV reactivation. Using the EBV P3HR-1 strain, we have reproduced in long-term culture of SVK epithelial cells an unusual pattern of infection previously observed in a subset of tumor biopsy samples: the persistence of het DNA in the absence of the parental helper virus. Fluorescence in situ hybridization (FISH) of infected cell subclones indicated the retention of het DNA in an integrated form. Incorporation of an intact het DNA molecule was confirmed by PCR, using primers that framed junctions of the four rearranged EBV DNA segments comprising P3HR-1-derived het DNA. Structural analysis of EBV terminal repeats revealed a banding pattern consistent with the integration of het DNA as a concatemer. Linkage of concatemeric monomers was defined at a nucleotide level, and that junctional sequence was detected in cell-free P3HR-1 virion DNA, confirming that subgenomic het DNA was packaged into infectious particles in a concatemeric configuration. Stable integration into cells having lost the standard viral genome allowed the unambiguous designation of het DNA as the source for viral gene products potentially encoded by both. Continuous expression of the latency-to-lytic switch protein Zta and detection of the BALF4 gene product gB, known to expand the target cell range of standard virus when incorporated at augmented levels into infectious progeny, add to a presumption of het DNA-enhanced pathogenesis in diseases of EBV reactivation.     

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.     

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.     

Virus-specific DNA in the cytoplasm of avian sarcoma virus-infected cells is a precursor to covalently closed circular viral DNA in the nucleus.

Three principal forms of viral DNA have been identified in cells infected with avian sarcoma virus: (i) a linear duplex molecule synthesized in the cytoplasm, (ii) a covalently closed circular molecule found in the nucleus, and (iii) proviral DNA covalently linked to high-molecular-weight cell DNA. To define precursor product relationships among these forms of viral DNA, we performed pulsechase experiments using 5-bromodeoxyuridine to label by density the linear species of viral DNA in the cytoplasm during the first 4 h after infection. After a 4-to 8-h chase with thymidine, a portion of the density-labeled viral DNA was transported to the nucleus and converted to a covalently closed circular form. We conclude that linear viral DNA, synthesized in the cytoplasm, is the precursor to closed circular DNA observed in the nucleus.     

Comparison of Epstein-Barr virus strains of different origin by analysis of the viral DNAs

Epstein-Barr virus (EBV) originating from Burkitt's lymphoma (P3HR-1 and CC34-5), nasopharyngeal carcinoma (M-ABA), transfusion mononucleosis (B95-8), and a patient with acute myeloblastic leukemia (QIMR-WIL) was isolated from virus-carrying lymphoid cell lines after induction with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Viral DNA was analyzed by partial denaturation mapping and by use of the restriction endonucleases EcoRI, HindIII, and SalI and separation of fragments in 0.4% agarose. By using the restriction enzyme data of B95-8 (EBV) and W91 (EBV) obtained by Given and Kieff (D. Given and E. Kieff, J. Virol. 28:524-542, 1978), maps were established for the other virus strains. Comigrating fragments were assumed to be identical or closely related among the different strains. Fragments of different strains migrating differently were isolated, purified, radioactively labeled, and mapped by hybridization against blots of separated viral fragments. The results were as follows. (i) All strains studied were closely related. (ii) The number of internal repeats was variable among and within viral strains. (iii) B95-8 (EBV) was the only strain with a large deletion of about 12,000 base pairs at the right-hand side of the molecule. At the same site, small deletions of about 400 to 500 base pairs were observed in P3HR-1 (EBV) and M-ABA (EBV) DNA. (iv) P3HR-1 (EBV), the only nontransforming EBV strain, had a deletion of about 3,000 to 4,000 base pairs in the long unique region adjacent to the internal repeats carrying a HindIII site. (v) Small inserted sequences of 150 to 400 base pairs were observed in M-ABA (EBV) and B95-8 (EBV) at identical sites in the middle of the long unique region. (vi) Near this site, an insertion of about 1,000 base pairs was found in P3HR-1 (EBV) DNA. (vii) The cleavage patterns of P3HR-1 virus DNA and the results of blot hybridizations with P3HR-1 virus fragments are not conclusive and point to the possibility that in addition to the normal cleavage pattern some viral sequences may be arranged differently. Even though it is possible that small differences in the genome organization may have significant biological effects, the great similarity among different EBV strains does not favor the hypothesis that disease-specific subtypes exist.     

Unintegrated viral sequences in rat cells transformed by simian virus 40 and a temperature sensitive A gene mutant

The status of viral sequences in rat cells transformed by simian virus 40 (SV40) and its temperature sensitive A gene mutant was investigated. Agarose gel electrophoresis of cell DNA prepared from clones picked from soft-agar and blot hybridization showed that sequences specific to SV40 genome were present both as integrated and unintegrated structures in rat clones. Digestion of rat cell DNA with various endonucleases with or without recognition sites in SV40 DNA and analysis indicated that the unintegrated viral genomes existed as full-length, covalently closed circular molecules. No differences in the free viral genomes were apparent between the clones transformed by the wild type and the mutant virus. The importance of the existence of free viral genomes in nonpermissive cells is discussed.     

Epstein-Barr virus with heterogeneous DNA disrupts latency.

By cloning the HR-1 Burkitt lymphoma line, we previously uncovered two distinct biological variants of nontransforming Epstein-Barr virus (EBV). The most commonly cloned variant has a low rate of spontaneous viral synthesis and is unable to induce early antigen in Raji cells (EAI-). A rare variant spontaneously releases virus which is capable of inducing early antigen in Raji cells (EAI+). Since EAI- virus lacks heterogeneous DNA (het-) and EAI+ virus contains heterogeneous DNA (het+), we suggested that spontaneous viral synthesis and induction of early antigen are biological properties which correlate with the presence of het sequences. The present experiments provide three new lines of experimental evidence in favor of this hypothesis. (i) Revertant subclones of the EAI+ het+ variant which have lost the het DNA concomitantly lost EAI ability. Thus, het DNA is not stably associated with the cells as are the episomes. (ii) het DNA was acquired by two het- subclones of the HR-1 line after superinfection with EAI+ virus. After superinfection, these clones synthesized EAI+ het+ virus. Thus, het DNA may be maintained in the HR-1 line by cell-to-cell spread. (iii) Virus with het DNA activated full expression of endogenous latent EBV of the transforming phenotype in a line of immortalized neonatal lymphocytes designated X50-7. By use of restriction endonuclease polymorphisms unique to both the superinfecting and endogenous genomes, we show that the genome of the activated virus resembles that of the virus which was endogenous to X50-7 cells. This result suggests that het sequences result in transactivation of the latent EBV. het DNA had homology with EBV sequences which are not normally contiguous on the physical map of the genome. het DNA was always accompanied by the presence of DNA of nonheterogenous HR-1. Thus, het DNA is a form of "defective" EBV DNA. However, the biological effect of this defective DNA is to enhance rather than to interfere with EBV replication. This is a novel property of defective virus.     

DNA of Epstein-Barr Virus. II. Comparison of the Molecular Weights of Restriction Endonuclease Fragments of the DNA of Epstein-Barr Virus Strains and Identification of End Fragments of the B95-8 Strain

Incubation of the DNA of the B95-8 strain of Epstein-Barr virus [EBV (B95-8) DNA] with EcoRI, Hsu I, Sal I, or Kpn I restriction endonuclease yielded 8 to 15 fragments separable on 0.4% agarose gels and ranging in molecular weight from less than 1 to more than 30 x 10(6). Bam I and Bgl II yielded fragments smaller than 11 x 10(6). Preincubation of EBV (B95-8) DNA with lambda exonuclease resulted in a decrease in the Hsu I A and Sal I A and D fragments, indicating that these fragments are positioned near termini. The electrophoretic profiles of the fragments produced by cleavage of the DNA of the B95-8, HR-1, and Jijoye strains of EBV were each distinctive. The molecular weights of some EcoRI, Hsu I, and Sal I fragments from the DNA of the HR-1 strain of EBV [EBV (HR-1) DNA] and of EcoRI fragments of the DNA of the Jijoye strain of EBV were identical to that of fragments produced by cleavage of EBV (B95-8) DNA with the same enzyme, whereas others were unique to each strain. Some Hsu I, EcoRI, and Sal I fragments of EBV (HR-1) DNA and Kpn I fragments of EBV (B95-8) DNA were present in half-molar abundance relative to the majority of the fragments. In these instances, the sum of the molecular weights of the fragments was in excess of 10(8), the known molecular weight of EBV (HR-1) and (B95-8) DNA. The simplest interpretation of this finding is that each EBV (HR-1), and possibly also (B95-8), DNA preparation contains two populations of DNA molecules that differ in the arrangement of DNA sequences about a single point, such as has been described for herpes simplex virus DNA. Minor fragments could also be observed if there were more than one difference in primary structure of the DNAs. The data do not exclude more extensive heterogeneity in primary structure of the DNA of the HR-1 strain. However, the observation that the relative molar abundance of major and minor fragments of EBV (HR-1) DNA did not vary between preparations from cultures that had been maintained separately for several years favors the former hypothesis over the latter.     

Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis.

A simple gel technique is described for the detection of large, covalently closed, circular DNA molecules in eucaryotic cells. The procedure is based on the electrophoretic technique of Eckhardt (T. Eckhardt, Plasmid 1:584-588, 1978) for detecting bacterial plasmids and has been modified for the detection of circular and linear extrachromosomal herpesvirus genomes in mammalian cells. Gentle lysis of suspended cells in the well of an agarose gel followed by high-voltage electrophoresis allows separation of extrachromosomal DNA from the bulk of cellular DNA. Circular viral DNA from cells which carry the genomes of Epstein-Barr virus, Herpesvirus saimiri, and Herpesvirus ateles can be detected in these gels as sharp bands which comigrate with bacterial plasmid DNA of 208 kilobases. Epstein-Barr virus producer cell lines also show a sharp band of linear 160-kilobase DNA. The kinetics of the appearance of this linear band after induction of viral replication after temperature shift parallels the known kinetics of Epstein-Barr virus production in these cell lines. Hybridization of DNA after transfer to filters shows that the circular and linear DNA bands are virus specific and that as little as 0.25 Epstein-Barr virus genome per cell can be detected. The technique is simple, rapid, and sensitive and requires relatively low amounts of cells (0.5 X 10(6) to 2.5 X 10(6)).     

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.     

Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line.

A non-integrated form of Epstein-Barr virus DNA was purified from the Burkitt lymphoma-derived human lymphoid cell line Raji by CsCl density gradient centrifugation and neutral glycerol gradient centrifugation. This intracellular form of the virus DNA sediments at a rate typical of a covalently closed circular DNA molecule of the size of the virus genome in both neutral and alkaline solution. Treatment with low doses of X-rays leads to a discontinuous conversion of the molecules to a form with the sedimentation properties of open circular DNA (a circular duplex molecule containing one or more single-strand breaks). The direct observation of large circular DNA molecules by electron microscopy further confirms the covalently closed circular duplex structure of part of the intracellular viral DNA. Such circular molecules were not detected in corresponding DNA fractions from Epstein-Barr virus-negative human lymphoid cell lines. In ethidium bromide/CsCl density gradient centrifugation experiments, the purified non-integrated virus DNA behaves as twisted, covalently closed DNA circles with the same initial superhelix density as polyoma virus DNA. The latter additional purification technique permits the isolation of intracellular Epstein-Barr virus DNA in > 90% pure form from non-producer cells. The molecular weight of the circular virus DNA from Raji cells, determined by contour length measurements, is the same within experimental error as that of the linear DNA from virus particles.     

DNA of Epstein-Barr virus. I. Comparative studies of the DNA of Epstein-Barr virus from HR-1 and B95-8 cells: size, structure, and relatedness.

We have compared the properties of the DNA of Epstein-Barr virus (EBV) purified from HR-1 (EBV HR-1 DNA) and B95-8 (EBV B95-8 DNA) continuous lymphoblast cultures. Our data indicate that (i) the S suc of native EBV DNA relative to T4D DNA is 55S. Using the modified Burgi-Hershey relationship (5), we estimate the molecular weight of native EBV DNA is 101 (plus or minus the molecular weight of native FBV DNA by measurement of the length of 3) times 106. Estimation of the molecule relative to form II PM2 DNA yields a value of 105 (plus or minus 3) times 106. (ii) After alkali denaturation, less than 50% of EBV DNA sediments as a single band in alkaline sucrose gradients in the region expected for DNA of 50 times 406 daltons. (iii) Intact EBV HR-1 and EBV B 95-8 DNAs band at 1.718 g/cm3 and a smaller band (approximately 25% of the DNA) AT 1.720 G/CM3. (IV) EBV HR-1 DNA possesses greater than 97% of the sequences of EBV B95-8 DNA. Hybrid DNA molecules formed between (3H)EBV HR-1 DNA and EBV HR-1 DNA or EBV B95-8 DNA had identical thermal stability. EBV B95-8 DNA lacks approximately 15% of the DNA sequences of EBV HR-1 DNA. We interpret these data to mean that EBV B95-8 is derived from a parental EBV through loss of genetic complexity. This defect may be linked to the ability of EBV B95-8 to "transform" lymphocytes invitro.