DNA of Epstein-Barr virus. IV. Linkage map of restriction enzyme fragments of the B95-8 and W91 strains of Epstein-Barr Virus.

The arrangement of EcoRI, Hsu I, and Sal I restriction enzyme sites in the DNA of the B95-8 and W91 isolates of Epstein-Barr virus (EBV) has been determined from the size of the single-enzyme-cleaved fragments and from blot hybridizations that identify which fragments cut from the DNA with one enzyme contain nucleotide sequences in common with fragments cut from the DNA with a second enzyme. The DNA of the B95-8 isolate was the prototype for this study. The data indicate that (i) approximately 95 X 10(6) to 100 X 10(6) daltons of EBV (B95-8) DNA is in a consistent and unique sequence arrangement. (ii) Both termini are variable in length. One end of the molecule after Hsu I endonuclease cleavage consists of approximately 3,000 base pairs, with as many as 10 additional 500-base pair segments. The opposite end of the molecule after Sal I endonuclease cleavage consists of approximately 1,500 base pairs, with as many as 10 additional 500-base pair segments. (iii) The opposite ends of the molecule contain homologous sequences. The high degree of homology between the opposite ends of the molecule and the similarity in size of the "additional" 500-base pair segments suggests that there are identical repeating units at both ends of the DNA. The arrangement of restriction endonuclease fragments of the DNA of the W91 isolate of EBV is similar to that of the B95-8 isolate and differs from the latter in the presence of approximately 7 X 10(6) daltons of "extra" DNA at a single site. Thus, the size of almost all EcoRI, Hsu I, and Sal I fragments of EBV (W91) DNA is identical to that of fragments of EBV (B95-8) DNA. A single EcoRI fragment, C, of EBV (W91) DNA is approximately 7 X 10(6) daltons larger than the corresponding EcoRI fragment of EBV (B95-8) DNA. Digestion of EBV (W91) DNA with Hsu I or Sal I restriction endonucleases produces two fragments (Hsu I D1 and D2 or Sal I G2 and G3) which differ in total size by approximately 7 X 10(6) daltons from the fragments of EBV (B95-8) DNA. Furthermore, the EcoRI, Hsu I, and Sal I fragments of EBV (W91) and (B95-8) DNAs, which are of similar molecular weight, have homologous nucleotide sequences. Moreover, the W91 fragments contain only sequences from a single region of the B95-8 genome. Two lines of evidence indicate that the "extra" sequences present in W91 EcoRI fragment C are viral DNA and not cellular. (i) The molecular weight of the "enlarged" EcoRI C fragment of EBV (W91) DNA is identical to that of the EcoRI C fragment of another isolate of EBV (Jijoye), (ii) The HR-1 clone of Jijoye has previously been shown to contain DNA which is not present in the B95-8 strain but is present in the EcoRI C and Hsu I D2 and D1 fragments of EBV (W91) DNA (N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388-398, 1978).     

Non-immortalizing P3J-HR-1 Epstein-Barr virus: a deletion mutant of its transforming parent, Jijoye.

The P3J-HR-1 strain of Epstein-Barr virus (EBV) fails to immortalize human lymphocytes. We wished to understand the nature of the genomic alterations which correlated with the loss of this ability. As a first step, the heterogeneity of DNA molecules in the P3J-HR-1 line was eliminated by cell cloning. Then a physical map was prepared of virion DNA from one cell clone, designated FF452-3. By comparison with the genomes of two EBVs, B95-8 and FF41, which are competent to immortalize lymphocytes, we identified a total of eight modifications of BamHI and EcoRI restriction endonuclease fragments of EBV (FF452-3) DNA consisting of insertions, deletions, or loss of a restriction endonuclease recognition site. To determine which of these alterations might be responsible for the loss of transforming phenotype, we examined homologous DNA fragments of the Jijoye strain of EBV, the progenitor of the HR-1 strain which still retains the ability to immortalize lymphocytes. We also studied viral DNA in lymphocytes transformed in vitro by Jijoye virus. Six of the eight alterations were found both in Jijoye and in clonal HR-1 DNA and were presumably genomic traits characteristic of this lineage of EBV. A small deletion in the BamHI-K fragment of HR-1 DNA was not found in Jijoye virion DNA, but this deletion was present in intracellular Jijoye DNA. Thus only one major genomic lesion in HR-1 DNA, a deletion of at least 2.4 x 10(6) molecular weight of DNA from a fused BamHI-H-Y fragment, consistently distinguished Jijoye DNA from its non-immortalizing P3J-HR-1 derivative. This deletion is likely to affect EBV genes which are directly or indirectly involved in immortalizing lymphocytes.     

DNA of Epstein-Barr virus. III. Identification of restriction enzyme fragments that contain DNA sequences which differ among strains of Epstein-Barr virus.

Previous kinetic and absorption hybridization experiments had demonstrated that the DNA of the B95-8 strain of Epstein-Barr virus was missing approximately 10% of the DNA sequences present in the DNA of the HR-1 strain (R.F. Pritchett, S.D. Hayward, and E. Kieff, J. Virol. 15:556-569, 1975; B. Sugder, W.C. Summers, and G. Klein, J. Virol. 18:765-775, 1976). The HR-1 strain differs from other laboratory strains, including the B95-8 and W91 strains, and from virus present in throat washings from patients with infectious mononucleosis in its inability to transform lymphocytes into lymphoblasts capable of long-term growth in culture (P. Gerber, Lancet i:1001, 1973; J. Menezes, W. Leibold, and G. Klein, Exp. Cell. Res. 92:478-484, 1975; G. Miller, D. Coope, J. Niederman, and J. Pagano, J. Virol. 18:1071-1080, 1976; G. Miller, J. Robinson, L. Heston, and M. Lipman, Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974). In the experiments reported here, the restriction enzyme fragments of Epstein-Barr virus DNA which contain sequences which differ among the HR-1, B95-8, and W91 strains have been identified. The DNA of the HR-1, B95-8, and W91 strains each differed in complexity. The sequences previously shown to be missing in the B95-8 strain were contained in the EcoRI-C and -D and Hsu I-E and -N fragments of the HR-1 strain and in the EcoRI-C and Hsu I-D and -E fragments of the W91 strain. The HR-1 strain was missing DNA contained in EcoRI fragments A and J through K and Hsu I fragment B of the B95-8 strain and in the EcoRI-A and Hsu I-B fragments of the W91 strain. The relationship of these data to the linkage map of restriction enzyme fragments of the DNA of the B95-8 and W91 strains (E. Kieff, N. Raab-Traub, D. Given, W. King, A.T. Powell, R. Pritchett, and T. Dambaugh, In F. Rapp and G. de-The, ed., Oncogenesis and Herpesviruses III, in press; D. Given and E. Kieff, submitted for publication) and the possible significance of the data are discussed.     

Epstein-Barr Virus-Specific RNA III. Mapping of DNA Encoding Viral RNA in Restringent Infection

Namalwa and Raji cells, originally obtained from a Burkitt tumor biopsy, grow as continuous cell lines in vitro and contain the Epstein-Barr virus (EBV)-related nuclear antigen EBNA (B. M. Reedman and G. Klein, Int. J. Cancer 11:499-520, 1973) and RNA homologous to at least 17 and 30% of the EBV genome, respectively (S. D. Hayward and E. Kieff, J. Virol. 18:518-525, 1976; T. Orellana and E. Kieff, J. Virol. 22:321-330, 1977). The polyribosomal and polyadenylated [poly(A)+] RNA fractions of Namalwa and Raji cells are enriched for a class of viral RNA homologous to 5 to 7% of EBV DNA (Hayward and Kieff, J. Virol. 18:518-525, 1976; Orellana and Kieff, J. Virol. 22:321-330, 1977). The objective of the experiments described in this communication was to determine the location within the map of the EBV genome (D. Given and E. Kieff, J. Virol. 28:524-542, 1978) of the DNA which encodes the viral RNA in the poly(A)+ and non-polyadenylated [poly(A)-] RNA fractions of Namalwa cells. Hybridization of labeled DNA homologous to Namalwa poly(A)+ or poly(A)- RNA to blots containing EcoRI, Hsu I, or Hsu I/EcoRI double-cut fragments of EBV (B95-8) or (W91) DNA indicated that these RNAs are encoded by DNA contained primarily in the Hsu I A/EcoRI A and Hsu I B/EcoRI A fragments and, to a lesser extent, in other fragments of the EBV genome. Hybridizations of Namalwa poly(A)+ and poly(A)- RNA in solution to denatured labeled EcoRI A or B fragments, Hsu I A, B, or D fragments, and Hsu I A/EcoRI A or Bam I S fragments and of Raji polyribosomal poly(A)+ RNA to the EcoRI A fragment indicated that (i) Namalwa poly(A)+ RNA is encoded primarily by 6 x 10(5) daltons of a 2 x 10(6)-dalton segment of DNA, Bam I S, which is tandemly reiterated, approximately 10 times, in the Hsu I A/EcoRI A fragment and is encoded to a lesser extent by DNA in the Hsu I B, EcoRI B, and Hsu I D fragments. Raji polyribosomal poly(A)+ RNA is encoded by a similar fraction of the EcoRI A fragment as that which encodes Namalwa poly(A)+ RNA. (ii) The fraction of the Bam I S fragment homologous to Namalwa poly(A)- RNA is similar to the fraction homologous to Namalwa poly(A)+ RNA. However, Namalwa poly(A)- RNA is homologous to a larger fraction of the DNA in the Hsu I B, Hsu I D, and EcoRI B fragments.     

Deletion of the Nontransforming Epstein-Barr Virus Strain P3HR-1 Causes Fusion of the Large Internal Repeat to the DSL Region

The nontransforming Epstein-Barr virus (EBV) strain P3HR-1 is known to have a deletion of sequences of the long unique region adjacent to the large internal repeats. The deleted region is believed to be required for initiation of transformation. To establish a more detailed map of the deletion in P3HR-1 virus, SalI-A of the transforming strain M-ABA and of P3HR-1 virus was cloned into the cosmid vector pHC79 and multiplied in Escherichia coli. The cleavage sites for BamHI, BglII, EcoRI, PstI, SacI, SacII, and XhoI were determined in the recombinant plasmid clones. Analysis of the boundary between large internal repeats and the long unique region showed that in M-ABA (EBV) the transition is different from that in B95-8 virus. The map established for SalI-A of P3HR-1 virus revealed that, in contrast to previous reports, the deletion has a size of 6.5 kilobase pairs. It involves the junction between large internal repeats and the long unique region and includes more than half of the rightmost large internal repeat. The site of the deletion in the long unique region is located between a SacI and a SacII site, about 200 base pairs apart from each other. The sequences neighboring the deletion in the long unique region showed homology to the nonrepeated sequences of the DS(R) (duplicated sequence, right) region. Sequences of the large internal repeat are thus fused to sequences of the DS(L) (duplicated sequence, left) region in P3HR-1 virus DNA under elimination of the DS(L) repeats. Jijoye, the parental Burkitt lymphoma cell line from which the P3HR-1 line is derived by single-cell cloning, is known to produce a transforming virus. Analysis of the Jijoye (EBV) genome with cloned M-ABA (EBV) probes specific for the sequences missing in P3HR-1 virus revealed that the sequences of M-ABA (EBV) BamHI-H2 are not represented in Jijoye (EBV). In Jijoye (EBV) the complete DS(L) region including the DS(L) repeats is, however, conserved. Further analysis of Jijoye (EBV) and of Jijoye virustransformed cell lines will be helpful to narrow down the region required for transformation.     

Cleavage of Epstein-Barr virus DNA by restriction endonucleases EcoRI, HindIII and BamI.

The cleavage of the DNAs of the B95-8 and P3HR-1 virus strains of Epstein-Barr virus by the restriction endonucleases EcoRI, HindIII and BamI was investigated using a new technique for quantitative evaluation of the fluorescence of ethidium stained DNA fragments separated on agarose gels. The results obtained with B95-8 DNA showed that in addition to the limited repetitions of nucleotide sequences observed in the EcoRI and HindIII cleavage patterns, the molecule contained a BamI fragment with a molecular mass of 2.0 megadaltons which was present in a total of about 11 copies and localized to a limited part of the DNA molecule. The same sequences were also present in the P3HR-1 DNA albeit in a lower molar ratio. P3HR-1 DNA yielded restriction enzyme cleavage patterns suggesting DNA sequence heterogeneity of P3HR-1 virus. No fragment was present in more than about 4 copies per molecule of P3HR-1 DNA. Comparison of the restriction enzyme cleavage patterns of P3HR-1 and B95-8 DNA revealed a high degree of structural homology emphasized by nucleic acid hybridization experiments with EBV complementary RNA synthesized in vitro.     

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.     

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.     

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.     

Organization of the Epstein-Barr virus DNA molecule. II. Fine mapping of the boundaries of the internal repeat cluster of B95-8 and identification of additional small tandem repeats adjacent to the HR-1 deletion.

We used cloned BamHI fragments from Epstein-Barr virus strain B95-8 [EBV(B95-8)]DNA to obtain detailed restriction maps of the region of the genome adjacent to the large internal repeat cluster. These maps together with the results of hybridization experiments using a 3.1-kilobase repeat probe defined more precisely the location of the injection between the internal repeat cluster and the flanking unique-sequence DNA. On one side (UL), the repeat sequences extended 600 +/- 80 base pairs (bp) into BamHI-Y; on the other side (US), they extended 1,300 +/- 200 bp into BamHI-C. Therefore, EBV(B95-8) DNA contained a nonintegral number of 3.1-kilobase repeat units, namely, 12.6 copies. The mapping studies also revealed a second series of internal tandem repetitions in EBV(B95-8) DNA located within the BamHI-H fragment. This cluster comprised 11 copies of a 135-bp repeat unit which contained a single site for the NotI restriction endonuclease. Hybridization to these cloned EBV(B95-8) fragments using total EBV(HR-1) DNA as probe indicated that the deletion in EBV(HR-1) removed all 3,000 bp of unique-sequence DNA which lay between the large 3.1-kilobase and the small 135-bp repeat clusters. Thus, the deletion which destroyed the transforming ability in the EBV(HR-1) virus was bounded on either side by tandem repetitions.     

Radiobiological Inactivation of Epstein-Barr Virus

Lymphocyte transforming properties of B95-8 strain Epstein-Barr virus (EBV) are very sensitive to inactivation by either UV or X irradiation. No dose of irradiation increases the transforming capacity of EBV. The X-ray dose needed for inactivation of EBV transformation (dose that results in 37% survival, 60,000 rads) is similar to the dose required for inactivation of plaque formation by herpes simplex virus type 1 (Fischer strain). Although herpes simplex virus is more sensitive than EBV to UV irradiation, this difference is most likely due to differences in the kinetics or mechanisms of repair of UV damage to the two viruses. The results lead to the hypothesis that a large part, or perhaps all, of the EBV genome is in some way needed to initiate transformation. The abilities of EBV to stimulate host cell DNA synthesis, to induce nuclear antigen, and to immortalize are inactivated in parallel. All clones of marmoset cells transformed by irradiated virus produce extracellular transforming virus. These findings suggest that the abilities of the virus to transform and to replicate complete progeny are inactivated together. The amounts of UV and X irradiation that inactivate transformation by B95-8 virus are less than the dose needed to inactivate early antigen induction by the nontransforming P(3)HR-1 strain of EBV. Based on radiobiological inactivation, 10 to 50% of the genome is needed for early antigen induction. Inactivation of early antigen induction is influenced by the cells in which the assay is performed. Inactivation proceeds more rapidly in EBV genome-free cells than in genome carrier Raji or in P(3)HR-1 converted EBV genome-free cells clone B(1). These results indicate that the resident EBV genome participates in the early antigen induction process. Variation in radio-biological killing of B95-8 and P(3)HR-1 EBV is not attributable to variations in the repair capacities of the cells in which the viruses were assayed, since inactivation of HSV was the same in primary lymphocytes and in all lymphoid cell lines tested.     

Genome of a mononucleosis Epstein-Barr virus contains DNA fragments previously regarded to be unique to Burkitt's lymphoma isolates

We wished to learn whether the genomes of strains of EMB isolated from patients with infectious mononucleosis are consistently distinguishable from those of strains from Burkitt's lymphoma. The genome of a new transforming strains (FF41) of EBV isolated from saliva of a patient with uncomplicated infectious mononucleosis was compared with the DNA of B95-8, the only other available virus from mononucleosis. It had been found previously that B95-8 has a deletion of about 8 Md in the region of the physical map represented by the Eco RI C, Hind III D, and Bam HI I fragments. The W91 and HR-1 isolates for Burkitt's lymphoma are not deleted in this region and it had been proposed that additional information was characteristic of EBV isolates from Burkitt's lymphoma. By means of restriction enzyme analysis, blot hybridization experiments and molecular cloning of FF41 DNA we demonstrate that the deletion found in B95-8 is not present in the new mononucleosis isolate. The FF41 genome contains an extra 8 Md of DNA, represented by Bam HI fragments B', W' and I', which are located in a larger Eco RI C fragment. Thus the genome of this salivary isolate contains DNA that had previously been regarded to be unique to strains from Burkitt's lymphoma. It is therefore unlikely that major insertions or deletions in the EBV genome account for differences in disease manifestation following EBV infection.     

Two strains of Epstein-Barr virus (B95-8 and a P3HR-1 subclone) that lack defective genomes induce early antigen and cause abortive infection of Raji cells.

The heterogeneity of Epstein-Barr virus (EBV) obtained from P3HR-1 cells has permitted derivation of a distinct subclone of P3HR-1 (L. Heston, M. Rabson, N. Brown, and G. Miller, Nature (London) 295:160-163, 1982). We have analyzed the biologic properties and genomic structure of this subclonal virus (clone 13) compared with those of parental P3HR-1 and B95-8 viruses. Synthesis of EBV compared with those of parental P3HR-1 and B95-8 viruses. Synthesis of EBV proteins in Raji cells superinfected with virus derived from P3HR-1, clone 13, and B95-8 was analyzed both by fluorography of radiolabeled proteins and by immunoblotting. Highly concentrated preparations of clone 13 and B95-8 virus induced most of the spectrum of EBV proteins in Raji cells with the exception of the 145,000-, 140,000-, and 110,000-molecular-weight proteins, which were either undetectable or reduced. Moreover, both clone 13 and B95-8 viruses also induced the same patterns of early antigen diffuse components as the parental P3HR-1 virus did. However, only P3HR-1 virus could induce EBV DNA synthesis in superinfected Raji cells, as determined both by buoyant density centrifugation and by in situ cytohybridization with biotinylated recombinant EBV DNA probes. Defective heterogeneous molecules present in P3HR-1 virus have been implicated in early antigen induction after superinfection of Raji cells. Therefore, Southern blots of clone 13, P3HR-1, and B95-8 viruses were hybridized to recombinant EBV fragments representing the sequences contained within the defective molecules in P3HR-1. The parental P3HR-1 contained the previously described defective molecules. No evidence for defective molecules was found in clone 13 or B95-8 viruses. These data indicate that concentrated preparations of both clone 13 and B95-8 viruses can induce abortive infection in Raji cells, but while the defective molecules are not needed for induction of early antigen diffuse components, they may be required for the induction of viral DNA synthesis.     

Expression of Epstein-Barr viral early antigen in monolayer tissue cultures after transfection with viral DNA and DNA fragments.

Antigens associated with the Epstein-Barr virus (EBV) replicative cycle were found in the nucleus and cytoplasm of human placental, Vero, BSC-1, and owl monkey kidney cells transfected with EBV DNA prepared from several different strains of virus. The number of antigen-positive nuclei increased when transfection was followed by cell fusion induced by inactivated Sendai virus. About 1,200 antigen-positive foci were induced per micrograms of EBV DNA. On the basis of their reactivity with various well-characterized human sera, it appears that the antigens are part of the early antigen complex. None of the four restriction endonucleases, EcoRI, HindIII, SalI, and BamHI, destroyed the ability of EBV DNA to induce early antigen. However, only SalI seemed to leave intact the full spectrum of antigen expression by the HR-1 and FF41 strains of EBV DNA. By means of transfection with recombinant DNA plasmids containing different EBV (FF41) DNA fragments regenerated by EcoRI, we showed that the coding region for early antigen was at least partially contained on the 17.2-megadalton EcoRI B fragment.     

Epstein-Barr virus DNA XII. A variable region of the Epstein-Barr virus genome is included in the P3HR-1 deletion.

The P3HR-1 subclone of Jijoye differs from Jijoye and from other Epstein-Barr virus (EBV)-infected cell lines in that the virus produced by P3HR-1 cultures lacks the ability to growth-transform normal B lymphocytes (Heston et al., Nature (London) 295:160-163, 1982; Miller et al., J. Virol. 18:1071-1080, 1976; Miller et al., Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974; Ragona et al., Virology 101:553-557, 1980). The P3HR-1 virus was known to be deleted for a region which encodes RNA in latently infected, growth-transformed cells (Bornkamm et al., J. Virol. 35:603-618, 1980; Heller et al., J. Virol. 38:632-648, 1981; King et al., J. Virol. 36:506-518, 1980; Raab-Traub et al., J. Virol. 27:388-398, 1978; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934, 1980). This deletion is now more precisely defined. The P3HR-1 genome contains less than 170 base pairs (and possibly none) of the 3,300-base pair U2 region of EBV DNA and is also lacking IR2 (a 123-base pair repeat which is the right boundary of U2). A surprising finding is that EBV isolates vary in part of the U2 region. Two transforming EB viruses, AG876 and Jijoye, are deleted for part of the U2 region including most or all of a fragment, HinfI-c, which encodes part of one of the three more abundant cytoplasmic polyadenylated RNAs of growth-transformed cells (King et al., J. Virol. 36:506-518, 1980; King et al., J. Virol. 38:649-660, 1981; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934).     

Palindromic structure and polypeptide expression of 36 kilobase pairs of heterogeneous Epstein-Barr virus (P3HR-1) DNA.

Among the Epstein-Barr virions (EBV) produced by the P3HR-1 (HR-1) cell line are a defective subpopulation with rearranged viral DNA designated heterogeneous DNA (het DNA). These defective virions are responsible for the capacity of HR-1 virus to induce early antigen in Raji c cells and for trans activation of latent EBV in X50-7 cells. Virions with het DNA are independent replicons which pass horizontally from cell to cell rather than being partitioned vertically. We analyzed the structure and defined several polypeptide products of het DNA to understand these remarkable biologic properties. A 36-kilobase-pair (kbp) stretch of het DNA was cloned (as two EcoRI fragments of 20 and 16 kbp) from virions released from a cellular subclone of HR-1 cells. The unusual aspect of the 20-kbp fragment was the linkage of sequences of BamHI-M and BamHI-B', which are not adjacent on the standard EBV genome. The 16-kbp fragment was a palindrome in which at least two additional recombinations on each side of the palindrome had linked regions of the standard EBV genome which are not normally contiguous. The 20-kbp het DNA fragment was attached to at least one and possibly both ends of the 16-kbp het DNA fragment. We identified antigenic polypeptides produced in COS-1 cells after gene transfer of various cloned het DNA fragments. The 20-kbp fragment encoded a cytoplasmic antigen of about 95 kilodaltons (kDa). The 16-kbp fragment encoded antigens located in the nucleus, nuclear membrane, and cytoplasm. These were represented by several polypeptides, the most prominent of which were about 55, 52, and 36 kDa. The 36-kDa polypeptide was localized to a 2.7-kbp BamHI fragment which had homology to standard BamHI-W and BamHI-Z. Another polypeptide of 50 kDa found in the nucleus was mapped to the 7.1-kbp BamHI het DNA fragment which spans the EcoRI site linking the 20- and 16-kbp fragments of het DNA. Thus, HR-1 het DNA encodes several discrete polypeptide products, one or more of which could be responsible for the unusual biologic properties of the virus. The composition, regulation, and ultimately the expression of some of these products relative to standard EBV is probably altered by the genomic rearrangements of het DNA.     

The structure of the termini of the DNA of Epstein-Barr virus.

We have studied the DNA of Epstein-Barr virus (EBV) isolated from the B95-8 strain of that virus (Miller and Lipman, 1973). When EBV DNA is partially digested with lambda-exonuclease and allowed to reanneal, up to 50% of the full-length molecules circularize. The arrangements of nucleotide sequences containing the terminal repeats identified in this circularization experiment have been determined. Those fragments of viral DNA generated by digestion with restriction endonucleases which are terminal and contain the terminal repeats have been identified by their sensitivity to digestion of full-length DNA by lambda-exonuclease and by virtue of their being partially homologous to one another. The population of DNA molecules in the B95-8 strain of EBV was found to be nonuniform. The nonuniformity results from different molecules having different numbers of a 0.37 megadalton terminal repeat at each end. About 70% of molecules have four terminal repeats at one end, while four equal classes, each comprising approximately 25% of the population, have one, two, three or four repeats at the other end. The arrangements of nucleotide sequences identified as being terminal in virion DNA were studied in the intracellular circular viral DNA of cells transformed by a single particle on EBV. All fragments produced by digestion with endonucleases and scored as being terminal in virion DNA were absent from intracellular circular DNA. An additional fragment was identified in the digests of intracellular DNA of each transformed clone. The molecular weights of the new fragments equal the sum of the molecular weights of two terminal fragments which are joined upon intracellular circularization of viral DNA.     

DNA of Epstein-Barr virus. VI. Mapping of the internal tandem reiteration.

Epstein-Barr virus (B95-8) DNA consists of short (10 X 10(6)) and long (87 X 10(6)) unique DNA sequences joined by 10 tandem reiterations of a 1.85 X 10(6) DNA segment. The reiterated sequence contains BamI and BglII sites separated by 4 X 10(5). The 4.5 X 10(5) and 14.0 X 10(5) segments generated by cleavage of the reiterated DNA with BamI and BglII contain sequences which hybridize to each other, suggesting that the internal tandemly reiterated sequence has a direct or inverted repeat within it. The opposite ends of the linear, nicked, double-stranded DNA molecule (R. F. Pritchett, S. D. Hayward, and E. D. Kieff, J. Virol. 15:556--569, 1975) consist of from 1 to 12 direct repeats of another 3 X 10(5) sequence (D. Given and E. Kieff, J. Virol. 28:524--542, 1978; D. Given, D. Yee, K. Griem, and E. Kieff, J. Virol. 30:852--862, 1979). There is no homology between the internal reiterated sequence and either terminus. However, part of the internal reiteration (less than 5 X 10(5) is reiterated at two separate locations in the long unique region. The internal reiterations are a source of variation within EBV (B95-8) DNA preparations. Thus, although the majority of molecules contain 10 tandem reiterations, some molecules have 9, 8, 7, 6, 5, 4, or fewer tandem reiterations. A consequence of this variability is that the KpnI A fragment and the EcoRI/Hsul A fragment consist of a family of seven or more fragments differing in the number of tandem internal reiterations. The EcoRI/HsuI A fragment of EBV (W91) DNA is approximately 6 X 10(6) smaller than the largest and dominant EcoRI/HsuI A fragment of EBV (B95-8) DNA. EBV (W91 DNA also differs from EBV (B95-8) DNA by an additional 7 X 10(6) to 8 X 10(6) of DNA in the long unique DNA region (D. Given and E. Kieff, J. Virol. 28:524--542, 1978; N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388--398, 1978). These data suggest the possibility that the smaller number of internal reiterations in EBV (W91) DNA may be a consequence of the additional unique DNA and a restriction in the overall size of EBV DNA.     

Sequences of the Epstein-Barr Virus (EBV) large internal repeat form the center of a 16-kilobase-pair palindrome of EBV (P3HR-1) heterogeneous DNA.

We have previously characterized several genomic rearrangements of Epstein-Barr virus (EBV) DNA contained in one of the defective EBV genomes harbored by the P3HR-1 (HR-1) line (H. B. Jenson, M. S. Rabson, and G. Miller, J. Virol. 58:475-486, 1986). One recombinant clone of heterogeneous DNA (het DNA) from this defective genome is an EcoRI fragment of 16 kilobase pairs (kbp) which is a palindrome. DNA digestion fragments specific for the center of this palindrome were present in cells which contained het DNA but not in cells which lacked het DNA. Thus, the palindrome was not an artifact of DNA cloning. The organization of the center of this palindrome was studied by DNA sequencing. The comparable region of the parental HR-1 genome was also studied by DNA sequencing. The central 3,495 base pairs (bp) of the palindrome were composed of sequences derived exclusively from internal repeat 1 of EBV, represented by BamHI W fragment. At each end of the central 3,495 hp was a symmetrical recombination with sequences of BamHI-Z, located more than 50 kbp away on the standard EBV genome. The central 3,495 bp were composed of an unduplicated 341 bp flanked by two perfect palindromic repeats of 1,577 bp. The 341-bp unique region was a portion of a 387-bp region of standard HR-1 BamHI-W which was identical to the central 387 bp of the palindrome. This central 387-bp region contained numerous stretches of dyad symmetry capable of forming a large stem-and-loop structure. The palindromic rearrangement had created two novel open reading frames in het DNA derived from standard HR-1 BamHI-W sequences. These two het DNA open reading frames had different amino termini but identical carboxy termini derived from the large open reading frame in standard HR-1 BamHI-W (HR-1 BWRF1). The BamHI-W sequences found in het DNA did not include either the TATA box of standard HR-1 BamHI-W or the exons which are present in the potentially polycistronic latent mRNAs encoding EBV nuclear antigens. These marked alterations in genomic structure may relate to the unique biologic properties of virus stocks containing het DNA by creation of new polypeptides or by formation or deletion of regulatory or functional signals.