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).     

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.     

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.     

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.     

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.     

DNA of Epstein-Barr virus. V. Direct repeats of the ends of Epstein-Barr virus DNA.

Previous data indicated that Epstein-Barr virus DNA is terminated at both ends by direct or inverted repeats of from 1 to 12 copies of a 3 X 10(5)-dalton sequence. Thus, restriction endonuclease fragments which include either terminus vary in size by 3 X 10(5)-dalton increments (D. Given and E. Kieff, J. Virol. 28:524--542, 1978; S. D. Hayward and E. Kieff, J. Virol. 23:421--429, 1977). Furthermore, defined fragments containing either terminus hybridize to each other (Given and Kieff, J. Virol. 28:524--542, 1978). The 5' ends of the DNA are susceptible to lambda exonuclease digestion (Hayward and Kieff, J. Virol. 23:421--429, 1977). To determine whether the terminal DNA is a direct or inverted repeat, the structures formed after denaturation and reannealing of the DNA from one terminus and after annealing of lambda exonuclease-treated DNA were examined in the electron microscope. The data were as follows. (i) No inverted repeats were detected within the SalI D or EcoRI D terminal fragments of Epstein-Barr virus DNA. The absence of "hairpin- or pan-handle-like" structures in denatured and partially reannealed preparations of the SalI D or EcoRI D fragment and the absence of repetitive hairpin- or pan-handle-like structures in the free 5' tails of DNA treated with lambda exonuclease indicate that there is no inverted repeat within the 3 X 10(5)-dalton terminal reiteration. (ii) Denatured SalI D or EcoRI D fragments reanneal to form circles ranging in size from 3 X 10(5) to 2.5 X 1O(6) daltons, indicating the presence of multiple direct repeats within this terminus. (iii) Lambda exonuclease treatment of the DNA extracted from virus that had accumulated in the extracellular fluid resulted in asynchronous digestion of ends and extensive internal digestion, probably a consequence of nicks and gaps in the DNA. Most full-length molecules, after 5 min of lambda exonuclease digestion, annealed to form circles, indicating that there exists a direct repeat at both ends of the DNA. (iv) The finding of several circularized molecules with small, largely double-strand circles at the juncture of the ends indicates that the direct repeat at both ends is directly repeated within each end. Hybridization between the direct repeats at the termini is likely to be the mechanism by which Epstein-Barr virus DNA circularizes within infected cells (T. Lindahl, A. Adams, G. Bjursell, G. W. Bornkamm, C. Kaschka-Dierich, and U. Jehn, J. Mol. Biol. 102:511-530, 1976).     

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.     

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.     

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 recombinants from overlapping cosmid fragments.

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)     

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).     

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.     

Epstein-Barr virus nuclear protein 2 is a critical determinant for tumor growth in SCID mice and for transformation in vitro.

Injection of Epstein-Barr virus (EBV)-transformed human lymphoblastoid B cells into immunodeficient SCID mice results in the appearance of rapidly growing, fatal human B-cell tumors. To evaluate the role of EBV nuclear protein 2 (EBNA-2) in this process, we generated lymphoblastoid cell lines transformed by several EBV mutants which were identical except for deletions in the EBNA-2 gene (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991). These cell lines were injected intraperitoneally into SCID mice, and the interval until tumor detection was determined. Cell lines transformed with EBV type 1 (strain W91) or with EBV type 2 (strain P3HR-1) with an inserted type 1 EBNA-2 gene grew at the same rapid rate, indicating the potential importance of EBNA-2 for tumor formation in vivo. Cell lines derived from three different EBV mutants with deletions in the amino half of EBNA-2 produced tumors more slowly than cell lines transformed by wild-type W91 virus. In contrast, a cell line transformed with an EBV mutant with a deletion in the carboxy terminus of EBNA-2 grew more rapidly than cell lines transformed by wild-type virus. EBV mutants with deletions in the amino half of EBNA-2 had had reduced transforming activity in vitro, while the carboxy-terminal EBNA-2 mutant had had transforming activity greater than or equal to that of the wild type. These data indicate that EBNA-2 plays a critical role both for B-cell tumor growth in SCID mice and for B-lymphocyte transformation in vitro.     

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.     

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.     

Heterogeneity of Epstein-Barr virus. III. Comparison of a transforming and a nontransforming virus by partial denaturation mapping of their DNAs.

The DNAs of a transforming and a nontransforming Epstein-Barr virus strain, B95-8 AND P3HR-1, were compared by partial denturation mapping. B95-8 viral DNA showed a homogeneous denaturation pattern. In contrast, P3HR-1 viral DNA was heterogeneous, containing at least two classes of molecules, classified into groups A and B and present in a ratio of about 2:1 to 3:1. No evidence could be obtained that molecules from both groups A and B contain identical sequences present in different orientations as described for herpes simplex viral DNA. The majority of sequences present in B95-8 and in P3HR-1 viral DNA group A could be correlated by assuming that different sequences, about 12,000 base pairs long, were inserted or deleted, respectively, at different position of both viral genomes.     

Cloning of Epstein-Barr virus (EBV) DNA fragments in pBR32 and Charon3A

Restriction fragments of Epstein-Barr virus (EBV; B95-8) DNA were cloned in the Tc gene of pBR322 (HindIII-F, -G, -I, -J, -K, -L, and -M) and in Charon3A (EcoRI-GI and -G2). Altogether these cloned fragments covered 39% of the entire viral genome. The cloned EcoRI-G2 fragment of EBV (B95-8) DNA was shown to contain, in addition to HindIII-J, two more HindIII-fragments : HindIII-M, which had not been located on the linkage map of the viral genome (Given and Kieff, 1978) and HindIII-N, which had been unrecognized up to now. The utility of this cloning method is discussed in regard to the detailed mapping of a viral genome and large-scale production of the viral DNA.     

Association of Epstein-Barr virus early antigen diffuse component and virus-specified DNA polymerase activity.

The role of Epstein-Barr virus (EBV) early antigen diffuse component (EA-D) and its relationship with EBV DNA polymerase in EBV genome-carrying cells are unclear, EBV-specified DNA polymerase was purified in a sequential manner from Raji cells treated with phorbol-12,13-dibutyrate and n-butyrate by phosphocellulose, DEAE-cellulose, double-stranded DNA-cellulose, and blue Sepharose column chromatography. Four polypeptides with molecular masses of 110,000, 100,000, 55,000, and 49,000 daltons were found to be associated with EBV-specified DNA polymerase activity. A monoclonal antibody which could neutralize the EBV DNA polymerase activity was prepared and found to recognize 55,000- and 49,000-dalton polypeptides. An EA-D monoclonal antibody, R3 (G. R. Pearson, V. Vorman, B. Chase, T. Sculley, M. Hummel, and E. Kieff, J. Virol. 47:183-201, 1983), was also able to recognize these same two polypeptides associated with EBV DNA polymerase activity. It was concluded that EBV EA-D polypeptides, as identified by R3 monoclonal antibody, are critical components of EBV DNA polymerase.     

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.     

Two families of sequences in the small RNA-encoding region of Epstein-Barr virus (EBV) correlate with EBV types A and B

DNA sequence analysis was carried out on the 1-kilobase SacI-EcoRI region of the EcoRI J fragment of four strains of Epstein-Barr virus (EBV) (MABA, P3HR-1, FF41, and NPC-5), and the sequences were compared with the prototype sequence from strain B95-8. Ten single-base changes which grouped the strains into two families (1 and 2) were found. Restriction endonuclease polymorphisms predicted from the sequences were used to classify the EBV DNA from a further 26 EBV-positive cell lines into these two families. The EBNA-2 types (A or B) of the strains were found to correlate with the J region type; EBNA-2 type A DNA regularly contained J region sequence type 1, while EBNA-2 type B DNA generally carried J region sequence type 2. These data are consistent with the notion of there being two distinct families of EBV with discrete, conserved differences in DNA sequence.