Sites of sequence variability in Epstein-Barr virus DNA from

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1 Proc. Nati. Acad. Sci. USA Vol. 76, No. 6, pp June 1979 C(ell Biology Sites of sequence variability in Epstein-Barr virus DNA from different sources (intracellular virus DNA/Burkitt lyntphoma/nasopharyngeal carcinoma/infectious mononucleosis) LARS RYMO, TOMAS LINDAHL, AND ALICE ADAMS Departments of Clinical Chemistry, Medical Chemistry, and Medical Microbiology, Cothenbhrg University' Communicated by George Klein, March 1, Gothenburg, Sveden ABSTRACT The intracellular Epstein-Barr virus (EBV) DNA present in virus-transformed cells was partly purified from 23 cell lines or biopsies of Burkitt lymphoma, nasopharyngeal carcinoma, infectious mononucleosis, or healthy carrier origin. Such DNA was cleaved in fragments (A-K) of molecular weights between 1 X 106 and 30 X 106 with restriction enzyme EcoRI, and these fragments were analyzed by standard methods involving agarose gel electrophoresis, transfer to nitrocellulose filters, and hybridization with radioactive EBV DNA or complementary RNA. Sequence variability among different EBV DNA isolates was largely confined to the A, C, and I fragments. These results are discussed in relation to the linkage map of the EcoRI fragments of EBV DNA. The EcoRI cleavage pattern of intracellular viral DNA of an EBV-like virus from baboon cells, Herpesvirus papio, was entirely different from that of human EBV isolates. Strain differences in tumor viruses have often been characterized by restriction enzyme cleavage of viral DNA followed by determinations of the sizes of the DNA fragments by gel electrophoresis (1). Since the Epstein-Barr virus (EBV) is associated with several distinctly different forms of human disease, it has been of interest to estimate the degree of strain variability of this virus. No useful experimental lytic system is available for production of EBV particles from many different sources. A small minority of virus-transformed cell lines spontaneously produce virus, and comparisons among Epstein-Barr virion DNAs have been limited to two or three different isolates (2-6). Human cell lines and tumor biopsies that contain EBV DNA without releasing virions nevertheless carry multiple copies of EBV DNA molecules, with both nonintegrated circular forms of viral genome length and sequence complexity and integrated viral DNA sequences being present (7, 8). In at least some cases, the virus may be rescued from such nonproducer cell lines by fusion with EBV-negative cells and induction (9). In the present study, nonintegrated EBV DNA molecules have been partly purified from lysates of cells originating from Burkitt lymphomas, poorly differentiated nasopharyngeal carcinomas, patients with infectious mononucleosis, or healthy carriers. This DNA has been fragmented and analyzed by standard methods involving cleavage by EcoRI enzyme, separation of the DNA fragments by gel electrophoresis, transfer to membrane filters, hybridization with radioactive EBV DNA or complementary RNA (crna), and fluorography (10-13). MATERIALS AND METHODS Cell Lines and Tumor Biopsies. Lymphoid cell lines were grown in stationary suspension culture. Cells from Kenyan tumor biopsies and tumor cells grown in nude mice were gifts from George Klein and were treated as described (14). Three Burkitt lymphoma biopsies (J.N., R.W., and A.G.) and two biopsies of poorly differentiated nasopharyngeal carcinoma (K.C. and L.L.) were investigated. The following cells known to contain circular nonintegrated EBV DNA were studied: the African Burkitt lymphoma lines Raji and Rael (8); the American Burkitt lymphoma line SU-AmB-2 (15); the nasopharyngeal carcinoma epithelial cell isolate M.M. grown in nude mice (14); the nasopharvngeal carcinoma lymphoblastoid cell line LY-28 (16); infectious mononucleosis lines Salomon, TW16, TW20, JHTC-33, IM-198, and cb35b1 (17); and lines F-265, NC-37, U-303 L, and SK-Li from individuals without EBV-associated disease (8, 16). In addition, the Kenyan Burkitt lymphoma line Daudi (18), the cbc29 line obtained by transformation of cord blood leukocytes with the B95-8 strain of EBV, and the EBVnegative lymphoma line U-698 (19) were investigated. Abongo is a Kenyan Burkitt lymphoma line grown in nude mice. The Herpesuirus papio-transformed baboon lymphoid cell line 18-C contains circular nonintegrated viral DNA of viral genome length* and was also included in the study. Partial Purification of Nonintegrated EBV DNA. High molecular weight DNA, about 10-fold enriched in EBV DNA, was prepared from cells as described (20). Briefly, cell lysates were obtained with Sarkosyl/EDTA, treated with Pronase, and fractionated by neutral CsCl density gradient centrifugation. DNA fractions of density g/cm3 were pooled, dialyzed free from Cs(-Il, and concentrated by ethanol precipitation. EB3V DNA preparations from virions released by B95-8 and P3HR-1 cells were made as described (20). Radioactive EBV DNA and crna. EBV DNA was labeled with 125I-labeled dctp (1251-dCTP) (21) to specific activities of 1-2 X 108 cpm/,ug by the nick-translation reaction of Escherichia coli DNA polymerase according to a procedure based on that of Rigby et al. (22) and Maniatis et al. (23). Carrier-free 1251-dCTP (approximately 109 cpm) dissolved in 50% ethanol was dried under reduced pressure and redissolved in 100 pl of 50mM Tris-HCI, ph 7.5/7 mm MgCI2/1 mm dithiothreitol/10 gm dctp/20 jm datp/20 pm dgtp/20 tm TTP. EB3V DNA (1 jig), 10 pg of DNase I (electrophoretically purified, Worthington), and 30 units of E. coli DNA polymerase (Boehringer Mannheim) were added, and the reaction mixture was incubated at 14WC for 3 hr. About 40% of the 1251 radioactivity became incorporated into trichloroacetic acid-precipitable material. The reaction wvas stopped by the addition of 100 y l of 0.1 M EDTA and the solution was extracted with equal volumes of phenol/cresol/water, 100:14:1 1 (containing 0.18 g of 8-hydroxyquinoline per liter), and chloroforin/isoamyl alcohol (100:1). The aqueous phase was chromatographed on a Sephadex C-50 column (0.5 X 10 cm) equilibrated with 10 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisemnent ' in accordance with 18 U. S. C solely to indicate this fact Abbreviations: EBV, Epstein-Barr virus; crna, complementary RNA. * 1.. Falk, 1'. Lindahl, G. Bjursell & G,. Klein, Int. J. Cancer, in press.

2 Cell Biology: Rymo et al. mm Tris-HC'l, ph 7.5/1 mm EDTA. The excluded fractions were pooled, heated to 1000C for 5 min, divided into portions, and stored frozen at -20'C. EBV crna was synthesized with P3HR-1 EBV DNA labeled with la-32plctp (New England Nuclear) and E. coli RNA polymerase (a gift from K. Carlsson, University of Tromso, Norway) as described (20). Analysis of Restriction Enzyme Cleavage Patterns. Partly purified EBV DNA (about 10 jig of total DNA containing ng of EBV DNA) in 200 Mil of 50 mm Tris-HCI, ph 7.5/100 mm NaCII/10 mm MgCl2/1 mm dithiothreitol was incubated for 2 hr at 370C with endonuclease EcoRI (Boehringer Mannheim). The amount of enzyme used was at least 5 times the amount needed for complete hydrolysis of an equivalent amount of adenovirus 2 DNA under the same conditions. The reaction was stopped by addition of 10,ul of 0.5 M EDTA, and the DNA was precipitated with ethanol at -20'C. The precipitate was dissolved in 50,ul of 20% glycerol/10 mm Tris-HCI, ph 7.5/0.1% sodium dodecyl sulfate/0.05% bromophenol blue, and DNA samples of 5-15 Mil were loaded into 5 X 2 X 4 mm slots in 26 X 16 X 0.5 cm horizontal 0.35% agarose (Litex HSC, Glostrup, Denmark) slab gels. The gels were run for 18 hr at 1.3 V/cm and 60C, using a buffer system containiig 50 mm Tris acetate (ph 7.9), 20 mm sodium acetate, 1 mm EDTA, and 0.5 jig of ethidium bromide per ml. B95-8 EBV DNA was included as a reference in all gels. The DNA in the gels was then denatured in situ and transferred to nitrocellulose sheets (BA85, Schleicher & Schuell) as described by Southern (10). After the filters were washed, dried, and baked at 80'"C for 4 hr, they were inserted into polyethylene bags and preincubated with Denhardt's solution (0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin) in quadruple strength NaCl/citrate (NaCl/citrate contains 0.15 M NaCl and 15 mm sodium citrate, ph 7.0)/0.1% sodium dodecyl stulfate/50 jig of sonicated calf thymus DNA per ml at 670C for 6 hr. Hybridization was carried out at 670C for 40 hr in 6 ml of Denhardt's solution containing quadruple strength NaCl/ citrate, 0.1% sodium dodecyl sulfate, 50 jig of sonicated DNA per ml, 5 mm potassium iodide, and 0.1 jig of '251-labeled nick-translated B95-8 EBV DNA. The membranes were rinsed in 2-fold concentrated NaCl/citrate and washed at 670C in the hybridization buffer for 3 hr, in 2-fold concentrated Na(CI/citrate/1% sodium dodecyl sulfate for 30 min, in NaCl/citrate for 30 min, in half strength NaCl/citrate for 30 min, and in quarter strength NaCII/citrate for 30 min. They were then air-dried and subjected to fluorography at -700C with Kodak X-Omat R film and Agfa Gevaert M.R.600 intensifying screens. Several different exposure times were needed to clearly discern both large and small fragments. Membranes to be hybridized with crna were not pretreated as described above for DNA/ DNA hybridization. Hybridization was carried out at 67' C for 40 hr in 6 ml of a solution containing quadruple strength Na(1/citrate. 0.1% sodium dodecyl sulfate, 0.1 mg of yeast UNA per tni, and 0.1 pg of 32P-labeled P3HRI-1 E13V crna. Th'le membranes were washed in 2-fold concentrated NaCl/ citrate at 370C for 2 hr, and then again at 67 'C for I hr, treated wvith 20 pg of RNase A per ml of 2-fold concentrated NaCl/ citrate at 37C' for 1 hr, arid washed with NaCI/citrate for several hours at room temperature. They were then dried arid fluorographed as described above. RESULTS Cleavage patterns analyzed with different EBV probes EBV DNA from virions released by B95-8 cells is cleaved byi EcoRl into 12 fragments (A-K) of Mrs betwveemi 30 X 106 and Proc. Natl. Acad. Sci. USA 76 (1979) X 106, and the total Mr of these fragments is close to 100 X 106, the Mr of intact EBV DNA (3, 5). Similar cleavage patterns to those of virion DNA were observed here with 23 different isolates of intracellular EBV DNA by the Southern blotting technique (Figs. 1 and 2). Linear EBV DNA is cleaved by EcoRI at one end adjacent to a short terminal redundancy (3), so circular viral DNA molecules, which are the predominant intracellular form studied here, would be expected to generate cleavage patterns similar to linear virion DNA. P3HR-1 virion DNA generated a more complex pattern (Fig. 2), in agreement with previous observations of heterogeneity within this strain (4, 5). It would appear that P3HR-1 is unique in this regard among the isolates investigated here. Two of the EcoRI fragments of EBV DNA, G1 and G2, are of very similar molecular weight and were not separated from each other by gel electrophoresis, and bands D and E were not well resolved in analyses by the blotting technique. With these minor reservations, all EBV DNA fragments in EcoRI digests of pure EBV DNA detected by optical methods were also detected here with either radioactive nick-translated B95-8 EBV DNA (Fig. 1) or EBV crna (Fig. 2) from the P3HR-1 strain as hybridization probes. No band was ever detected that could be visualized with only one probe. These observations strongly indicate that the crna is an adequate probe representative of most (or all) EBV DNA sequences, although abundance differences among various sequences exist. Thus, the smaller I and J bands were consistently stronger than the H band when crna was used as the probe (Fig. 2) A- B- *W C DE - F G H s w w~ Jo- * K v. UP Fl(;. 1. E(oRl cleavage patterns of partly purified, nonintegrated circular EBV DNA fromn virus-transf)rmied cells. The DNA fragments were separated by electrophoresis in 0.35% agarose gels, denatured iln situ, transferred to nitrocellulose membrane filters, incubated with EWV '251-labeled I)NA (1'2'l-DNA). and analyzed by fluorography. Partly pqrified intracellular EBV DNA (2 pg) was applied to each slot. Slots: 1, B95-8 virion DNA (100 ng); 2, B95-8 virion DNA (10 ng); 3, LL. nasop)haryngeal carcinoma tumor biopsy; 4, K.C. nasopharyngeal carcinoma tumor biopsy; 5, A.0. Burkitt lymphoma tumor biopsy; 6, R.W. Burkitt lymphomia tumor biopsy; 7, B95-8 virion DNA (10 ng): 8,.JH'l'C-33 (infectious mononucleosis cell line); 9. Salomon (infectious mononucleosis cell line); 10, (2 Pg of DNA from an EBV-negative lymphoma): 11, virion DNA (10 ng); 12. blank; 13..I.N. Burkitt lymphoma tumor l)iopsy; 14, Rael (Burkitt lymphoma line); 15. B95-8 virion D)NA (10 ng).

3 2796 Cell Biology: Rymo et al. Proc. Natl. Acad. Sci. USA 76 (1979) U( CO ^ 4g A.. t.i~ D E *,a 4w -.w 4w t" 0 io. 0,ft 0 Go F-- J "4. ~~~~~~~~~~~~~~~~~~~I I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.- Ki- A E3 FI(. 2. EcoRI cleavage patterns of intracellular EBV DNA. Conditions were as in Fig. 1, except that EBV [32P~cRNA was used as the probe instead of 1251-D)NA. In the left half of the gel, about 3 pg of total DNA was applied to each slot, whereas in the right half, 1.5,ug of DNA of the same material was analyzed. Slots: 1 and 9, P3HR-1 virion DNA (10 ng); 2, 10, and 17, B95-8 virion DNA (10 ng); 3 and 11, Raji (Burkitt lymphoma line); 4 and 12, TW16 (infectious mononucleosis line); 5 and 13, TrW20 (infectious mononucleosis line); 6 and 14, NC-37 (line from carrier without lymphoproliferative disease); 7 and 15, cbc 29 (cord blood cells immortalized with B95-8 EBV); 8 and 16, StT-AmB-2 (American Burkitt lymphoma line). Strain differences By comparison of the EcoRI cleavage patterns of many different EBV DNA isolates, it became clear that the sizes of certain fragments show considerable variation, whereas other fragments appear highly invariant. In the latter group, the F, H, and J fragments had the same size in all isolates, and the (D+E) and (G1+G2) bands were also always present. The B band was of the same size in 24 of 25 isolates, the exception being EBV DNA from LY-28 cells (Fig. 3). The LY-28 line is derived from a Hong Kong nasopharyngeal carcinoma biopsy (24) and is the only Asian EBV strain studied here. In material from LY-28, the B fragment was missing but two additional bands were instead found close to the smaller D band, suggesting that the B fragment in LY-28 EBV DNA contains an extra EcoRl cleavage site. A similar sequence alteration was not found in EBV DNA of African nasopharyngeal carcinoma isolates (Fig. 1). The largest fragment, A, which is mainly comprised of multiple copies of a repeated fragment of Mr 2 X 106 (5) varied markedly in size among different isolates. B95-8 virion DNA yielded all A fragment of Mr about 29 X 106 containing about 11 copies of the reiterated sequence. A fragments of similar size were found in EBV DNA from Raji, Daudi, Abongo, Su- AmB-2, Mbane, LY-28, NC-37, F-265, 8&3-1, and U-303L cells. Smaller A fragments of MrS between 23 X 106 and 26 X 106 were observed with material from all five tumor biopsies investigated as well as the Rael, IM-198, Salomon, TW16, TW20, JHTC-33, cb35l31, cbc29, and SK-LI lines. A small A fragment of this type was also observed for the dominating sequence of P3HR-1 virion DNA (Fig. 2). Many differences are found in the C fragment region, and this section of the EBV DNA sequence shows the greatest variability among different isolates. The C fragment has a Mr of about 11.4 X 106 in the B95-8 virion DNA and in material from the cb,35bl and SK-LI lines and the A.O. and K.C. tumor biopsies, but it may instead be entirely absent (TW16, TW20, 883-L), present as an anomalously small fragment of Af r about.11 D FE 10 W. G Fic.. 3. EcoRI cleavage patterns of EBV DNA from IM-198 (infectious mononucleosis) and LY-28 (nasopharyngeal carcinoma). 9.7 X 106 (L.L. tumor biopsy, Mbane, Salomon, cbc29), or present as a large fragment of Mr about 13.0 X 106 (tumor biopsy R.W., LY-28, IM-198, JHTC-33, U-303L). Moreover, several EBV isolates show two bands between the B and (D+E) bands, apparently corresponding to two of the sizes found with individual C fragments. Thus, EBV DNA from the J.N. tumor biopsy, Raji, F-265, and NC-37 yielded two bands of Mr about 13.0 X 106 and 11.4 X 106, whereas material from Rael, Daudi, Abongo, and SU-AmB-2 yielded two bands of Mr 13.0 X 106 and 9.7 X 106. No EBV DNA isolate gave two bands of Mr 11.4 X 106 and 9.7 X 106, however. We note that in one batch of virion DNA from B95-8 cells, two C, bands, apparently in molar amounts, occurred with a larger component Mr 13.0 X 106 present in addition to the C fragment normally found (Fig. 1). This extra band had been observed in two earlier B95-8 EBV DNA preparations as a submolar fragment and was not a result of partial cleavage of the EBV DNA with the EcoRI enzyme, because it was present after digestion for various times with several concentrations of different EcoRI preparations. Moreover, no C band at all was observed with EBV DNA from 883-L cells, although the B95-8 EBV strain is derived from 883-1L (25). A minority of the EBV DNA isolates were missing the I band, whereas the smaller J band could be clearly visualized in the same digests. Among 25 different isolates, the I fragment was not found in EBV D)NA from the A.O. tumor biopsy. Mbane, R.!!W..

4 Cell Biology: IM-198, JHTC-33, Salomon, SK-L1, and U-303L. Although some of the lines used in this study produce small quantities of EBV particles, none of the lines missing the I fragment produce detectable amounts of virions. Finally, a few EBV DNA isolates showed an extra band between the F and G bands. The DNA/DNA hybridization data indicated that this band was present in submolar amounts, and its presence suggests minor heterogeneity within the particular EBV DNA isolate. It was not detected in any of the EBV DNA preparations from tumor biopsies, but it was reproducibly observed in material from the Raji, Daudi, Rael, Mbane, LY-28, NC-37, and F-265 lines. DNA sequence differences between EBV and an EBVlike virus from baboons Baboons have a herpes virus carried in B lymphocytes which has been termed Herpesvirus papio, and DNA from H. papio contains sequences cross-hybridizing with human EBV (26). Circular nonintegrated H. papio DNA has been found in a virus-transformed baboon lymphoid cell line, 18-C,* and partly purified H. papio DNA from this line was cleaved with EcoRI and analyzed with an EBV DNA probe (Fig. 4). A heavy band was found between the B and C' bands of the B95-8 EBV DNA reference, and five weaker bands between the A and B, E and F, F and G, and J and K bands were also detected. There was no similarity between the band patterns generated from any of the EBV DNA isolates and H. papio DNA, suggesting that H. papio is a distinct virus only distantly related to EBV. A- c G H- RN-mo et al. ILO Ao lb Proc. Natl. Acad. Sci. USA 76 (1979) 2797 DISCUSSION In this comparison between the EcoRI fragment patterns of 23 different isolates of circular EBV DNA molecules from cells and 2 virion DNAs, several types of variation confined to certain regions of the virus genome were detected. In particular, the A and C' fragments were highly variable in size, whereas the C' or I fragments were missing in a minority of the isolates without appearance of new detectable bands. On the other hand, within the sensitivity of the technique used, the B, (D+E), F, (Gt+G2), H, and J fragments appeared to be generally conserved. A linkage map of the EcoRI fragments of B95-8 EBV DNA has recently become available (3). This linkage map is shown in Fig. 5. in which the repeated sequences within the A fragment and the terminal redundant regions are also indicated. The arrows under the map show the two locations of sequence heterogeneity observed between B95-8 EBV DNA and the major component in P3HR-1 EBV DNA by partial denaturation mapping (4). These two DNAs differ with regard to the sizes of the isolated EcoRI A and C fragments, in apparent agreement with the partial denaturation mapping data. In the many additional EBV DNA isolates investigated here, size differences in the A and (C fragments remain the most common variations. 'T'he blotting method used in this work only allows a semiquantitative estimate of the abundance of each fragment, so luplications of certain parts of the virus genome to preserve its length after deletion of sequences would be difficult to detect. For example it has been shown by optical methods that P3HR-1 EBV DNA has two copies of the I-J region but fewer repeat sequences in the A region than B95-8 EBV DNA (5). The present method would hardly detect the former alteration while registering the latter one as a change in the size of the A band. Further, the loss of one of the overlapping D and E bands would not be detected. For such reasons, it is not possible at this point to correlate differences between the cleavage patterns of various isolates with a total genomne size difference. Several lymphoid cell lines transformed with B95-8 virus in vitro contain EBV DNA circles of 10-15% reduced size (17, 27). These molecules do not lack any EcoRI fragment entirely but have a shorter A fragment thams the virion DNA used for immortalization. It is not known if this is the whole explanation of the DNA circle size variation in this case. Although the differences in size of the A fragment among isolates from many sources may well depend on a variation in the number of repeat sequences within this fragmnent. the marked heterogeneity of the C fragment is more (lifficult to rationalize. A more limited sequence repeat region could perhaps occur also within this fragment. Alternatively, although isolated EBV DNA appears free from detectable amounts of cellular DNA sequences (2), the possibility that a sniall, variable stretch of host DNA may be included in the C fragnient cannot presently be ruled out. J Il 11 A G G F K B E H 11 II C D a ~~~~~~ = J - FIG. 4. EcoRI cleavage patterns of human EBV DNA (B95-8 virion DNA) and Herpesuirus papito )NA (from 18-( baboon cell line) I I I I I I I I I I I I x106 FIu;. 5. Linkage map of 'E(olI fragments of B95-8 EBV DNA (3). The sites of endonuclease cleavage are indicated by perpendicular lines above the open bar denioting the linear B95-8 DNA molecule. The lines within the open bar indicate the repeated sequences present in the A fragment and the terminal redundant regions. The lower scale denotes size in terms of molecular weight of double-stranded I)NA.

5 2798 Cell Biology: Rymo et al. The different EBV DNA isolates investigated here were largely similar, and in most cases only one or two differences were detected among individual cleavage patterns. In four separate cases, the patterns were even indistinguishable. Interestingly, the American Burkitt lymphoma line SU-AmB-2 yielded EBV DNA circles with the same EcoRI cleavage pattern as EBV DNA from the African Burkitt lymphoma Abongo, propagated in nude mice. These EBV DNA preparations were made and analyzed at different times, precluding the possibility of crosscontamination. The structural similarity between intracellular EBV DNA from biopsies and cell lines has been noted in a previous study on a smaller number of isolates by similar techniques (13). Because no attempt was made to purify the nonintegrated EBV DNA in that work and the high agarose concentration in the gels precluded an adequate size estimation of the largest DNA fragments, it is difficult to compare those results with the present ones in more detail. It has often been speculated that viral strain differences, in addition to cellular immunologic and genetic factors may be important for the disparate interactions of EBV with its host. However, at the present level of resolution, no obvious disease-related differences could be discerned among EBV DNA isolates of either tumor or nontumor origin. While the present approach essentially rules out that highly defective EBV strains are associated with certain diseases, more subtle sequence differences within the EBV genome may well still be of critical biological importance. Studies of viral transcription products in different types of transformed cells or direct sequencing of certain interesting regions of EBV DNA isolates may provide more decisive answers with regard to the eventual importance of EBV strain variability. Note Added in Proof. EBV DNA from the widely used lines Raji, NC-37, and F-265 show identical EcoRI cleavage patterns in this paper. Additional studies have shown that the HindIll cleavage patterns of these EBV DNAs also are identical, and isozyme patterns and other properties of the original isolates of these lines strongly indicate that NC-37 and F-265 are sublines of Raji rather than lines established from normal carriers. Thus, the present data for the EBV DNA from NC-37 and F-265 should not be regarded as representative of material from normal cells (unpublished results). We thank George Klein and Goran Magnusson for discussions and Jane Ohnander, Berit Sperens, and Loraine Karran for technical assistance. This work was supported by U.S. Public Health Service Contract NO1-CP within the Virus Cancer Program of the National Cancer Institute and by the Swedish Cancer Society. Proc. Natl. Acad. Sci. USA 76 (1979) 1. Nathans, D. & Danna, K. J. (1972) J. Mol. Biol. 64, Raab-Traub, N., Pritchett, R. & Kieff, E. (1978) J. Virol. 27, Given, D. & Kieff, E. (1978) J. Virol. 28, Delius, H. & Bornkamm, G. W. (1978) J. Virol. 27, Rymo, L. & Forsblom, S. (1978) Nucleic Acids Res. 5, Sugden, B., Summers, W. C. & Klein, G. (1976) J. Virol. 18, Nonoyama, M. & Pagano, J. S. (1973) Nature (London) 242, Kaschka-Dierich, C., Falk, L., Bjursell, G., Adams, A. & Lindahl, T. (1977) Int. J. Cancer 20, Glaser, R. & Nonoyama, M. (1974) J. Virol. 14, Southern, E. M. (1975) J. Mol. Biol. 98, Ketner, G. & Kelly, T. J. (1976) Proc. Natl. Acad. Sci. USA 73, Botchan, M., Topp, W. & Sambrook, J. (1976) Cell 9, Sugden, B. (1977) Proc. Natl. Acad. Sci. USA 74, Kaschka-Dierich, C., Adams, A., Lindahl, T., Bornkamm, G. W., Bjursell, G., Klein, G., Giovanella, B. C. & Singh, S. (1976) Nature (London) 260, Koliais, S., Bjursell, G., Adams, A., Lindahl, T. & Klein, G. (1978) J. Natl. Cancer Inst. 60, Adams, A. (1979) in The Epstein-Barr Virus, eds. Epstein, M. A. & Achong, Y. M. (Springer, Berlin), in press. 17. Adams, A., Bjursell, G., Gussander, E., Koliais, S., Falk, L. & Lindahl, T. (1979) J. Virol. 29, Klein, G. & Dombos, L. (1973) Int. J. Cancer 11, Klein, G., Lindahl, T., Jondal, M., Leibold, W., Menezes, J., Nilsson, K. & Sundstrom, C. (1974) Proc. Natl. Acad. Sci. USA 71, Lindahl, T., Adams, A., Bjursell, G., Bornkamm, G. W., Kaschka-Dierich, C. & Jehn, U. (1976) J. Mol. Biol. 102, Scherberg, N. H. & Refetoff, S. (1974) Biochim. Biophys. Acta 340, Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) J. Mol. Biol. 113, Maniatis, T., Jeffery, A. & Kleid, D. G. (1975) Proc. Natl. Acad. Sci. USA 72, de The, G., Ho, H. C., Kwan, H. C., Desgranges, C. & Favre, M. C. (1970) Int. J. Cancer 6, Miller, G. & Lipman, M. (1973) Proc. Natl. Acad. Sci. USA 70, Falk, L., Deinhardt, F., Nonoyama, M., Wolfe, L. G., Bergholz, C., Lapin, B., Yakovleva, L., Agrba, V., Henle, G. & Henle, W. (1976) Int. J. Cancer 18, Adams, A., Bjursell, G., Kaschka-Dierich, C. & Lindahl, T. (1977) J. Virol. 22,