Identification of transcribed regions of Epstein-Barr virus DNA in Burkitt lymphoma-derived cells.

RNA was extracted from the Burkitt lymphoma-derived cell line Raji and from Burkitt lymphoma tumor biopsies, isotope labeled in vitro by iodination with 125I, and hybridized to electrophoretically separated restriction endonuclease fragments of Epstein-Barr virus DNA on nitrocellulose membranes. The results indicated that only certain parts of the Epstein-Barr virus genome are represented as polyribosomal RNA in Raji cells, with a pronounced dominance of RNA sequences complementary to a 2.0 x 10(6)-dalton segment of Epstein-Barr virus DNA located close to the left end of the viral genome. A map of virus-specific polyribosomal RNA sequences was constructed, which indicated that a minimum of three regions of the Epstein-Barr virus genome are expressed in Raji cells. Total-cell RNA preparations from five Burkitt lymphoma biopsies contained RNA sequences homologous to the same regions of Epstein-Barr virus DNA as polyribosomal RNA from Raji cells, albeit at different relative proportions.     

De novo DNA methylation at nonrandom founder sites 5' from an unmethylated minimal origin of DNA replication in latent Epstein-Barr virus genomes

Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells.     

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.     

X-linked lymphoproliferative disease: a karyotype analysis

X-linked lymphoproliferative disease (XLP) is triggered by Epstein-Barr virus. This virus is also associated with Burkitt lymphoma, a tumor that carries specific chromosome translocations. No such chromosome translocations have been observed in an analysis of XLP-derived cell lines. One XLP patient was found also to have Klinefelter syndrome, having inherited two copies of his maternal XLP-carrying X chromosome.     

Specific sites for EBV association in the Namalwa Burkitt lymphoma cell line and in a lymphoblastoid line transformed in vitro with EBV

Localization of Epstein-Barr virus (EBV) DNA was studied by in situ hybridization on chromosomes from the Namalwa Burkitt lymphoma cell line and from a lymphoblastoid cell line transformed in vitro (ATL9/g). The five chromosome bands 1p32, 1q31, 5q21, 13q21, and 16p13 showed the presence of EBV DNA in both of the lines. Grain deposition at the site on chromosome 1q of the Burkitt line was particularly intense. It was also found that EBV DNA in the lymphoblastoid cell line co-localized with a stable achromatic gap at 1p32 whose presence seems to confer a proliferative advantage on the cells.     

Directed integration of minute virus of mice DNA into episomes.

Recent studies with adeno-associated virus (AAV) have shown that site-specific integration is directed by DNA sequence motifs that are present in both the viral replication origin and the chromosomal preintegration DNA and that specify binding and nicking sites for the viral regulatory Rep protein. This finding raised the question as to whether other parvovirus regulatory proteins might direct site-specific recombination with DNA targets that contain origin sequences functionally equivalent to those described for AAV. To investigate this question, active and inactive forms of the minute virus of mice (MVM) 3' replication origin, derived from a replicative-form dimer-bridge intermediate, were propagated in an Epstein-Barr virus-based shuttle vector which replicates as an episome in a cell-cycle-dependent manner in mammalian cells. Upon MVM infection of these cells, the infecting genome integrated into episomes containing the active-origin sequence reported to be efficiently nicked by the MVM regulatory protein NS1. In contrast, MVM did not integrate into episomes containing either the inactive form of the origin sequence reported to be inefficiently nicked by NS1 or the active form from which the NS1 consensus nick site had been deleted. The structure of the cloned MVM episomal recombinants displayed several features previously described for AAV episomal and chromosomal recombinants. The findings indicate that the rules which govern AAV site-specific recombination also apply to MVM and suggest that site-specific chromosomal insertions may be achievable with different autonomous parvovirus replicator proteins which recognize binding and nicking sites on the target DNA.     

Integrated viral DNA sequences in Epstein-Barr virus-converted human lymphoma lines.

Most human lymphoid cell lines contain multiple copies of circular, nonintegrated Epstein-Barr virus (EBV) DNA molecules as well as viral DNA sequences with properties of integrated DNA. The physical state of the EBV DNA in a human lymphoma line that only contains one virus genome equivalent per cell has now been studied by three different methods, neutral CsCl density gradient centrifugation, actinomycin D-CsCl gradient centrifugation, and Hirt fractionation. This cell line, AW-Ramos, has been obtained by EBV infection in vitro of the apparently EBV-negative Ramos lymphoma line. The results indicate that the EBV DNA in AW-Ramos is present exclusively in a linearly integrated form. Similar data were obtained with two other EBV-converted sublines of Ramos cells.     

Recombinant junctions formed by site-specific integration of adeno-associated virus into an episome.

A model system using an episomal Epstein-Barr virus shuttle vector was recently developed to study the adeno-associated virus (AAV) site-specific integration event in chromosome 19q13.3-qter (C. Giraud, E. Winocour, and K.I. Berns, Proc. Natl. Acad. Sci. USA 91:10039-10043, 1994). In this study, we analyze the recombinant junctions generated after integration of the AAV genome into an Epstein-Barr virus shuttle vector carrying 8.2, 1.6, or 0.51 kb of the chromosome 19 preintegration sequence (AAVS1 locus). In most of the recombinants, one end of the viral genome was joined to a portion of the AAVS1 DNA previously shown to be a minimum target for AAV integration. Within this AAVS1 segment, the AAV insertion points were strikingly clustered around a binding site for the AAV regulatory protein. In all cases, the second junction with AAV occurred with vector DNA outside of the AAVS1 segment. With respect to the viral genome, one junction with the shuttle vector DNA occurred either within the AAV inverted terminal repeat (itr), or near the P5 promoter, approximately 100 nucleotides distal to a modified itr. The modified itr in 5 of 11 recombinants involved a head-to-tail organization. In one such instance, the AAV insert contained slightly more than one genome equivalent arranged in a head-to-tail manner with a junction close to the P5 promoter; the AAV insert in this recombinant episome could be rescued by adenovirus infection and replicated to virus particles. The significance of the head-to-tail organization is discussed in terms of the possible circularization of AAV DNA before or during integration.     

Epstein-Barr virus RNA in Burkitt tumor tissue.

Analysis of the viral RNA in four Burkitt tumor biopsies indicates that tumor tissue contains RNA homologous to at least 3–6% of the DNA of Epstein-Barr virus (EBV). Most of these RNA species accumulate in the polyadenylated RNA fraction of Burkitt tumor tissue. Two approaches have been used to determine the location within the EBV genome of the DNA sequences which encode stable RNA in two Burkitt tumor biopsies, F and S, which contain 6–10 copies per cell of at least 80% of the EBV genome. With the first approach, ³²P-EBV DNA homologous to polyadenylated or nonpolyadenylated RNAs from the F, S or R tumors was hybridized to blots of fragments of EBV DNA. With the second approach, polyadenylated or nonpolyadenylated RNAs from the F or S tumors were hybridized to separated, labeled fragments of EBV DNA in solution. The results indicate that first, most of the viral RNA in Burkitt tumor tissue is encoded by approximately 20% of the Hsu I D fragment, 20% of the Eco RI A/Hsu I A double-cut fragment and 3% of the Hsu I B fragment of EBV DNA; second, an abundant RNA species in tumor tissue is homologous to the “additional DNA” present in the W91 and Jijoye/HR-I Burkitt tumor isolates of EBV and absent in the B95-8 virus, an isolate of EBV from outside the Burkitt endemic region; and third, there is little or no homology to other regions of the EBV genome.     

When Epstein-Barr virus persistently infects B-cell lines, it frequently integrates.

In this study we used Gardella gel analysis of intact DNA, Southern blotting of digested DNA, and fluorescence in situ hybridization to provide complementary and unequivocal information on the state of the Epstein-Barr virus (EBV) genome in persistently infected cells. The fluorescence in situ hybridization technique allowed us to directly visualize both integrated and episomal EBV DNA at the single-cell level. We show here that circularization of the EBV genome is rarely detected upon infecting activated normal B cells. The virus can persist upon infection of a different proliferating B-cell target, EBV-negative Burkitt's lymphoma tumor cell lines. Analysis of 16 such lines reveal again, that the virus infrequently persists as covalently closed episomes; rather, the virus preferentially persists by integrating into the host DNA (10 of 16 clones). The integrated virus is linear and usually intact, although 3 of 10 isolates have deletions from the left-hand end including the latent origin of replication. At the level of our analysis, no obvious relationship was seen between the integration sites. These studies provide, for the first time, a reproducible in vitro model system to study integration by EBV.     

Synethesis and integration of viral DNA in chicken cells at different time after infection with various multiplicities of avian oncornavirus.

To see if integration of the provirus resulting from RNA tumor virus infection is limited to specific sites in the cell DNA, the variation in the number of copies of virus-specific DNA produced and integrated in chicken embryo fibroblasts after RAV-2 infection with different multiplicities has been determined at short times, long times, and several transfers after infection. The number of copies of viral DNA in cells was determined by initial hybridization kinetics of single-stranded viral complementary DNA with a moderate excess of cell DNA. The approach took into account the different sizes of cell DNA and complementary DNA in the hybridization mixture. It was found that uninfected chicken embryo fibroblasts have approximately seven copies, part haploid genome of DNA sequences homologous to part of the Rous-association virus 2 (RAV-2) genome. Infection with RAV-2 adds additional copies, and different sequences, of RAV -2- specific DNA. By 13 h postinfection, there are 3 to 10 additional copies per haploid genome. This number can not be increased by increasing the multiplicity of infection, and stays relatively constant up to 20 h postinfection, when some of the additional viral DNA is integrated. Between 20 and 40 h postinfection, the cells accumulated up to 100 copies per haploid genome of viral DNA. Most of these are unintegrated. This number decreases with cell transfer, until cells are left with one to three copies of additional viral DNA sequences per haploid genome, of which most are integrated. The finding that viral infection causes the permanent addition of one to three copies of integrated viral DNA, despite the cells being confronted with up to 100 copies per haploid genome after infection, is consistent with a hypothesis that chicken cells contain a limited number of specific integration sites for the oncornavirus genome.     

Epstein-Barr virus DNA is amplified in transformed lymphocytes.

Leukocytes isolated from two adult donors who lacked detectable antibodies to antigens associated with Epstein-Barr virus were exposed to an average of 0.02 to 0.1 DNA-containing particles of Epstein-Barr virus per cell and immediately clones in agarose. Within about 30 generations all transformed cell clones contained between 5 and 800 copies of viral DNA per cell. Only 1 in 10(4) to less than 1 in 10(5) of the cells of each clone release virus, and the frequency of release did not correlate with the average number of copies of viral DNA in the cells of each clone. One clone that had an average of five copies of viral DNA per cell was recloned, and the average number of copies in four of six subclones increased 15-to 50-fold while the subclones were being propagated sufficiently to study them. These results indicate that Epstein-Barr virus DNA can undergo amplification relative to cell DNA at different times after it transforms cells.     

Detection of the latent form of Epstein-Barr virus DNA in the peripheral blood of healthy individuals.

Epstein-Barr virus infects resting B cells in vitro and activates them to continuously proliferating lymphoblasts. Activation is essential for the virus to convert its linear genome to the covalently closed circular episomal form in which it persists in proliferating cells. However, in vivo, Epstein-Barr virus persists in resting B cells. We found that in these cells also the virus is present as an episome, suggesting that the cells must, at some time, have been activated and then returned to a resting state. This is the first direct demonstration, for any herpesvirus, of this form of the viral genome in normal persistently infected tissue. Since no linear viral DNA was detected, we estimate that fewer than 1 in 40 cells replicates the virus in the peripheral blood of healthy donors.     

Epstein-barr virus regulates c-MYC, apoptosis, and tumorigenicity in Burkitt lymphoma

Loss of the Epstein-Barr virus (EBV) genome from Akata Burkitt lymphoma (BL) cells is coincident with a loss of malignant phenotype, despite the fact that Akata and other EBV-positive BL cells express a restricted set of EBV gene products (type I latency) that are not known to overtly affect cell growth. Here we demonstrate that reestablishment of type I latency in EBV-negative Akata cells restores tumorigenicity and that tumorigenic potential correlates with an increased resistance to apoptosis under growth-limiting conditions. The antiapoptotic effect of EBV was associated with a higher level of Bcl-2 expression and an EBV-dependent decrease in steady-state levels of c-MYC protein. Although the EBV EBNA-1 protein is expressed in all EBV-associated tumors and is reported to have oncogenic potential, enforced expression of EBNA-1 alone in EBV-negative Akata cells failed to restore tumorigenicity or EBV-dependent down-regulation of c-MYC. These data provide direct evidence that EBV contributes to the tumorigenic potential of Burkitt lymphoma and suggest a novel model whereby a restricted latency program of EBV promotes B-cell survival, and thus virus persistence within an immune host, by selectively targeting the expression of c-MYC.     

A novel autonomously replicating sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-1.

Several autonomously replicating sequences of Hansenula polymorpha DL-1 (HARSs) with the characteristics of tandem integration were cloned by an enrichment procedure and analyzed for their functional elements to elucidate the mechanism of multiple integration in tandem repeats. All plasmids harboring newly cloned HARSs showed a high frequency of transformation and were maintained episomally before stabilization. After stabilization, the transforming DNA was stably integrated into the chromosome. HARS36 was selected for its high efficiency of transformation and tendency for integration. Several tandemly repeated copies of the transforming plasmid containing HARS36 (pCE36) integrated into the vicinity of the chromosomal end. Bal 31 digestion of the total DNA from the integrants followed by Southern blotting generated progressive shortening of the hybridization signal, indicating the telomeric localization of the transforming plasmids on the chromosome. The minimum region of HARS36 required for its HARS activity was analyzed by deletion analyses. Three important regions, A, B, and C, for episomal replication and integration were detected. Analysis of the DNA sequences of regions A and B required for the episomal replication revealed that region A contained several AT-rich sequences that showed sequence homology with the ARS core consensus sequence of Saccharomyces cerevisiae. Region B contained two directly repeated sequences which were predicted to form a bent DNA structure. Deletion of the AT-rich core in region A resulted in a complete loss of ARS activity, and deletion of the repeated sequences in region B greatly reduced the stability of the transforming plasmid and resulted in retarded cell growth. Region C was required for the facilitated chromosomal integration of transforming plasmids.     

Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line

Here we describe development and application of highly sensitive fluorescence methodology for localization of single-copy sequences in interphase nuclei and metaphase chromosomes by nonisotopic in situ hybridization. Application of this methodology to the investigation of Epstein-Barr virus integration in the Namalwa lymphoma cell line has revealed two EBV genomes closely integrated at the known site on chromosome 1. Detecting sequences as small as 5 kb, we further demonstrate resolution within interphase nuclei of two fragments of the viral genome spaced only 130 kb apart. Results indicate that the viral genomes are in opposite orientations and separated by roughly 340 kb of cellular DNA. This work demonstrates the feasibility and resolving power of interphase chromatin mapping to assess the proximity of closely spaced DNA sequences. Implications for virology, gene mapping, and investigation of nuclear organization are discussed.