Proc. Nat. Acad. Sci. USAVol. 72, No. 4, pp. 1477-1481, April 1975

Epstein-Barr Virus Genomes with Properties of Circular DNA Molecules inCarrier Cells

(virus latency/herpesviruses/Raji cells)

ALICE ADAMS AND TOMAS LINDAHL

Departments of Tumor Biology and Chemistry, Karolinska Institutet, Stockholm 60, Sweden

Communicated by George Klein, December 24, 1974

ABSTRACT A high-density fraction ofhigh-molecular-weight DNA was isolated from the human lymphoidcell line Raji. This cell line contains 50 to 60 virus genomeequivalents of Epstein-Barr virus DNA per cell, and thehigh-density DNA fraction was 10-fold enriched in suchviral sequences. Sedimentation analysis on neutralglycerol gradients, followed by hybridization experimentswith viral complementary RNA, showed that most of theintracellular viral DNA sequences in this material did notcosediment with the cellular DNA, but were recovered astwo distinct species with sedimentation coefficients of100 S and 65 S. These two forms sediment 1.70-1.75 and1.10-1.12 times as fast as the linear Epstein-Barr virusDNA from virus particles, and thus have the hydrodynamicproperties of a covalently closed circular form and anicked (containing single-strand breaks) circular formof the virus genome. The lOOS form also behaved as acovalently closed circular EBV DNA molecule on gradientcentrifugation in CsCl/propidium diiodide. It wouldappear that latent Epstein-Barr virus DNA has the proper-ties of a mammalian episome, and that both noninte-grated and integrated viral DNA sequences can be isolatedfrom carrier cells.

Herpesvirus latency differs in several respects from the trans-formed states induced by smaller DNA viruses in heterologousmammalian cells. Multiple complete copies of the herpesvirusgenome are apparently present in the carrier cells both in vivoand in cell lines grown in vitro (1). With the Epstein-Barr virus(EBV), immortalization of human lymphocytes can be in-duced at a very high frequency in vitro with presumably non-defective virus particles (2). Further, the structural relation-ship between the virus and the host genome is in part differentfrom that observed with other mammalian tumor viruses(3, 4).The established human lymphoid cell lines that carry

the EBV genome provide a good system to study herpesviruslatency in vitro. Most studies on the intracellular state ofEBV DNA have been performed with the Raji cell line, whichwas established in culture from a Burkitt lymphoma biopsy (5).Raji cells contain 50-60 EBV genome equivalents per cell inlatent form (6). We have previously shown that DNA "jointmolecules" comprised of both cellular and viral DNA se-quences can be isolated from Raji cells (7), and that at leastpart of this association between viral DNA and cellular DNAis due to alkali-stable bonds (8). These data indicate thatsome integrated virus DNA sequences are present, but it isnot known if these integrated sequences represent com-plete virus genomes or fragments of virus DNA. In the pres-

ent work, we report that nonintegrated EBV DNA sequencesare also present in Raji cells, in confirmation of the previousfindings of Nonoyama and Pagano (3), and that the non-inte-grated EBV genomes apparently have a circular conformation.

MATERIALS AND METHODS

Reagents. [3H]Thymidine (21 Ci/mmol) was obtained fromNew England Nuclear Chemicals. Pronase and propidiumdiiodide were purchased from Calbiochem. The Pronase waspreincubated as a 1% solution in 50 mM Tris * HCl (pH 7.5)for 2 hr at 370 before use. Reagent grade glycerol was dilutedwith an equal volume of water and passed through a mem-brane filter before use.

DNA Preparations. Raji cells were grown as suspensioncultures at 370 in RPMI 1640 medium supplemented with10% fetal bovine serum. For radioactive labeling of theDNA, cultures were diluted to a density of 2 X 105 cells perml, and 30-40 hr later [3H]thymidine (0.25 gCi/ml) wasadded. After an additional 40 hr of incubation, the cells wereharvested and washed twice with sodium phosphate-bufferedsaline. The cells were then suspended at a concentration of107 cells per ml in phosphate-buffered saline and lysed by theaddition of 0.5 volume of 3% Sarkosyl (Geigy) in 75 mMTris HCl/25 mM EDTA (pH 9.0). After gentle rolling toobtain a clear, viscous lysate, 0.1 volume of 1% Pronase wasadded, and the mixture was incubated for 2 hr at 37°. Thesolution was then cooled to room temperature (210) anddiluted with 15 volumes of deaerated 50 mM Tris HCl (pH8.5). After 1 hr of slow swirling to obtain a homogeneous solu-tion containing 5,/g/ml of DNA, solid CsCl was added anddissolved in the viscous DNA solution. In some experiments,the DNA solution was further treated with phenol as de-scribed (7), followed by dialysis, prior to addition of the CsCl.EBV [3H]DNA (7 X 104 cpm/ug) was isolated from virus

particles recovered from the P3HR-1 cell line as described (7).['4C]Thymidine-Jabeled DNA (2 X 104 cpm/ug) from Kleb-siella pneumoniae was prepared by the same methods as forEscherichia coli DNA (9). Phage T4 [14C]DNA (1.4 X 104cpm//Ag) and phage PM2 [32P]DNA (104 cpm//Ag) were pre-pared by standard methods.

Gradient Centrifugations. High-molecular-weight Raji [3H]-DNA was fractionated on neutral CsCl gradients as described(7). Briefly, the Raji DNA in CsCl solution was supplementedwith a trace quantity (10 ng/ml) of sheared K. pneumoniae[14C]DNA (p = 1.717 g/cm3) as a reference density markerand centrifuged for 65 hr at 33 000 rpm and 20°. Forty-five

Abbreviations: EBV, Epstein-Barr virus; cRNA, RNA com-plementary to DNA; EDTA, ethylenediaminetetraacetate.

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1478 Microbiology: Adams and Lindahl Proc. Nat. Acad. Sci. USA 72 (1975)

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FIG. 1. Cosedimentation of EBV [3H]DNA and phage T4['4C] DNA on a neutral glycerol gradient. The gradient (10-30%glycerol, 37 ml) contained 1 M NaCl/0.02 M Tris HCl/1 mMEDTA (pH 8.0) and was centrifuged at 25,000 rpm for 5 hr at210 in a Spinco SW 27 rotor. One milliliter fractions were col-lected from the bottom of the gradient and analyzed for 'H and14C radioactivity. A- -A, EBV DNA; 0-- -0, T4 DNA.

0.4 ml fractions were collected through a large hole in thebottom of the tube. Aliquots of each fraction were removedfor determination of 'H and 14C radioactivity and ability tohybridize with [32P]RNA complementary to EBV DNA(EBV cRNA).

Fractions to be further analyzed on neutral glycerol gradi-ents were pooled, and phage T4 [14C]DNA was added as a

reference. The CsCl was removed by dialysis against 0.1 MNaCl/0.02 M Tris- HCI/1 mAM EDTA (pH 8.0) for 4 hr at 2°,followed by packing the dialysis bag in solid, finely groundpolyethylene glycol 6000 for 1 hr to reduce the liquid volumeapproximately 4-fold, and further dialysis for 16 hr. Onemilliliter aliquots of DNA solutions (6 Mg/ml) were appliedwith wide-mouth pasteur pipettes on top of 37 ml glycerolgradients.For further analysis of the DNA recovered from glycerol

gradients on CsCl/propidium diiodide gradients (10), ap-propriate fractions were pooled and freed from glycerol bydialysis against 1 AVI NaCl/0.02 M Tris HCl/1 mnM EDTA(pH 8.0) for 20 hr at 20, followed by dialysis against 0.05 MINaCl/0.02 Tris HCl/1 mM EDTA (pH 8.0) for 3 hr, con-

centration with polyethylene glycol as described above, andfurther dialysis against the last buffer for 3 hr. To 2.2 mlaliquots of the DNA solution (0.12 jg/ml) in 5 ml centrifugetubes, 0.2 ml of 0.6% propidium diiodide in 0.01 M Tris * HCl(pH 8.0) and 2.25 g of solid CsCl were added. The CsCl was

dissolved by gentle swirling, and the tubes were overlayeredwith paraffin oil and centrifuged at 40,000 rpm and 200 in a

Spinco type SW 50.1 rotor for 48 hr. After centrifugation,thirty 0.1 ml fractions were collected from the bottom of eachtube, and the propidium diiodide was removed by extractingeach fraction three times with 3 volumes of 90% isopropanol.Twenty microliters of each fraction were then removed todetermine 'H radioactivity, and the remaining material was

used for nucleic acid hybridization experiments.

Nucleic Acid Hybridization. The preparation of EBV ["2P]cRNA and the hybridization procedures have been described(7, 11). To each gradient fraction containing DNA to beanalyzed, one volume of 1 AM NaOH and 0-0.005 volume of0.1% salmon sperm DNA were added, to obtain an alkali-denatured DNA solution containing a total of 5 /g of DNA.The solutions were then heated at 800 for 10 min to destroy

any large covalently closed DNA circular structures thatmight be present, cooled to 200, brought to pH 7.5, and passedthrough membrane filters.

Immunofluorescence. Assays for EBV-induced "early anti-gen" were performed according to Klein et al. (12) on cellsmears from each culture.

RESULTS

Sedimentation Properties ofEBV DNA from Virus Particles.EBV ['H]DNA (30 ng) from virus particles produced by theBurkitt-lymphoma-derived cell line P3HR-1 (13) was mixedwith phage T4 ['4C]DNA (100 ng) and analyzed by a sedi-mentation velocity experiment in a neutral glycerol gradientcontaining 1 M Na+ (Fig. 1). As previously reported (14),EBV DNA sediments slightly more slowly than T4 DNA, at0.94-0.95 times the rate of the phage DNA. The same resultwas also obtained on cosedimentation of the EBV DNA and T4DNA with an excess (6 ug) of high-molecular-weight, non-radioactive Raji DNA. The latter conditions were chosento simulate those present when the intrinsic EBV DNAsequences in Raji cells were analyzed (see below).The size and hydrodynamic properties of T4 DNA have

been studied in great detail. We use the mean values fromseveral investigations as calculated by Freifelder (15): T4DNA has a sedimentation coefficient of 61.8 S, and a molecularweight of 1.1 X 108. EBV DNA then has a sedimentationcoefficient of 58-59 S, and a molecular weight of 1.0 X 108,assuming a linear double-stranded structure.

Isolation of a High-Density DNA Fraction from Raji Cells.The Raji cultures employed were actively growing in amedium containing 0.25 /ACi/ml of ['H ]thymidine, and at thetime of harvest contained >90% living cells. Cell smears wereprepared from all cultures at the time of harvest and screenedfor "early antigen"-positive cells. No such cells were found(<0.005% positive cells). As the expression of early antigenprecedes viral DNA synthesis in the abortive lytic viruscycle (16), the EBV DNA characterized here should be repre-sentative of the latent virus genomes of nonproducer cells.The direct characterization of the sedimentation properties

of the intracellular EBV DNA sequences present in Raji cellsis difficult because only very small quantities of large DNAcan be analyzed on a glycerol or sucrose gradient. We there-fore performed a preliminary purification step to enrich forthe EBV DNA sequences present in high-molecular-weight(o- 1.5 X 108) DNA prepared from Raji cells. Due to the highguanine - cytosine content of the virus DNA, EBV DNA can beseparated from the bulk of the cellular DNA by isopycnicbanding in CsCl (7). Fig. 2 shows a typical fractionation of60 /ug of Raji DNA on an 18 ml CsCl gradient. Approxi-mately 80% of the total'EBV DNA applied to the gradientwas recovered in the high-density region, with a peak at adensity of 1.716 g/cm'. It should be emphasized that eventhough the majority of the EBV DNA sequences in Raji cellsband at a density close to that of free virus DNA (p = 1.718g/cm'), this in itself does not prove that nonintegrated EBVDNA sequences are present (7). Because of the similarity insize of the EBV genome and the DNA fragments being ana-lyzed, the theoretical distribution (17) of EBV DNA sequencesover such gradients will be very similar (though not identical)for the two extreme cases of either 100% integrated or 100%nonintegrated viral genomes.

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Epstein-Barr Virus DNA in Carrier Cells 1479

Properties of EBV DNA in Carrier Cells. The high-density['H]DNA fraction, which was 10-fold enriched in EBV DNAsequences as compared to unfractionated Raji DNA, wasmixed with a trace quantity (0.2 ,gg) of T4 [14C]DNA as sizereference, and analyzed by sedimentation through neutralglycerol gradients. The bulk of the cellular ['HIDNA sedi-mented as a broad peak at 70 S (Fig. 3). It may be predictedthat if all the intracellular EBV DNA were integrated, theEBV DNA sequences localized by nucleic acid hybridizationof individual gradient fractions should cosediment with thecellular DNA. On the other hand, if Raji cells contain freeEBV DNA molecules with the same sedimentation proper-ties as EBV DNA from virus particles, a peak of EBV DNAshould be present at 58-59 S. As can be seen from Fig. 3,neither of these predictions is correct. Instead, the majorityof the EBV DNA sequences are found as two distinct peakswith unique sedimentation coefficients. The larger of, thesepeaks has a sedimentation coefficient of 65 S, and thus sedi-ments more slowly than most of the cellular DNA, but morerapidly than T4 DNA. The smaller peak migrates consider-ably more rapidly than the bulk of the DNA on the gradientand has a sedimentation coefficient of 100 S. These two EBVDNA forms, therefore, sediment 1.10-1.12 and 1.70-1.75times faster than the EBV DNA molecules present in virusparticles. As circular forms of DNA are known to sedimentfaster than a.corresponding linear DNA molecule (18), thesedata indicate that the 100S form is a covalently closed EBVDNA circle and the 65S form a nicked EBV DNA circle.For comparison, it is noted that the covalently closed circularDNA and nicked circular DNA forms of phage P1 recoveredfrom lysogenic bacteria sediment 1.8 versus 1.09 times fasterthan the linear DNA molecule from P1 phage particles (19).

Properties of 1OOS Form. A more direct test for covalentlyclosed circular DNA molecules is based on the restrictedintercalation of dye molecules such as ethidium bromide orpropidium diiodide in theDNA, causing covalently closedDNAcircles to have a higher density in dye/CsCl gradients thanother DNA conformations (10). Fractions 10 to 14, containingthe 100S EBV DNA from two glycerol gradients run inparallel with the one shown in Fig. 3, were pooled and analyzedon two CsCl/propidium diiodide gradients. In a separatecontrol experiment, covalently closed circular ["P]DNAfrom phage PM2 was mixed with ['H]DNA from Raji cellsand analyzed in the same fashion. When Raji ['H]DNA fromthe 95 to 110S region of the glycerol gradients was analyzed,8-10% of the total DNA banded as a small peak at the posi-tion of covalently closed circular DNA, with the remainingDNA banding as linear, or nicked circular DNA (Fig. 4).The small DNA peak was greatly enriched in EBV DNAsequences, and contained approximately 50% of the total EBVDNA recovered from the gradient. The remaining EBV DNAbanded together with the bulk of the ['H]DNA. The latterEBV DNA fraction may have represented circular moleculesthat had acquired one or more single-strand breaks due to 'Hdecay and/or nuclease action during the recovery and analysisof the material from the glycerol gradients.The peak of ['H]DNA at the position of covalently closed

circular DNA in Fig. 4 is to a considerable extent due to EBVDNA. Of the total DNA in Raji cells, 0.10-0.15% consists ofEBV DNA (6). Here, a 10-fold purification of EBV DNAsequences was achieved in the initial CsCl gradient frac-tionation, followed by an additional 5- to 6-fold purification on

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FIG. 2. Enrichment for EBV DNA sequences from Raji cellDNA by density gradient centrifugation in neutral CsCl. Aliquotsof 18 ml each of a solution of Raji [3H]DNA (4 ,ug/ml) and K.pneumoniae ["CC]DNA (10 ng/ml) in CsCl were centrifuged for65 hr at 33,000 rpm in a Spinco type 60 Ti rotor at 200. At theend of the run, the 3H and 14C radioactivity and the ability tohybridize with EBV [32P]cRNA were determined on aliquotsfrom each fraction. The nonspecific background radioactivity(500 cpm) with salmon DNA in corresponding CsCl solutionshas been subtracted from the hybridization data. The arrowshows the position of the peak of the density marker, K. pneu-moniae DNA (, = 1.717 g/cm3). 0 0, Raji [3H]DNA;A- -A, EBV cRNA hybridized. Fractions 20 to 23 were pooledand used for further experiments.

isolation of the 100S DNA from the glycerol gradients. Afinal 3- to 4-fold purification was obtained in the dye/CsClgradient. From the specific activity of the ['H ]thymidine-labeled DNA (correcting for the difference in base compositionbetween EBV DNA and cellular DNA) and the amount ofEBV ["IP]cRNA hybridized, it is estimated that this peak

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FIG. 3. Fractionation of high-density DNA from Raji cellson a neutral glycerol gradient. High-density [3H]DNA (6 Mug)recovered from CsCl gradients (Fig. 2) was mixed with T4[14C]DNA (0.2 Mg) and centrifuged on a glycerol gradient. Theexperimental conditions were as in Fig. 1, except that the time ofcentrifugation was only 3 hr. For each fraction, the 'H and 14Cradioactivity and the ability to hybridize with EBV [32P]cRNAwere determined. The arrow shows the position of the size marker,T4 [14C]DNA. The fractions 32 to 35 contained the 14C densitymarker used in the CsCl gradients, which was well separatedfrom the T4 ['4C]DNA. 0*, Raji [3H]DNA;A--A, EBVcRNA hybridized.

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1480 Microbiology: Adams and Lindahl

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FIG. 4. Density-gradient centrifugation in CsCl/propidiumdiiodide gradients of a fast-sedimenting fraction (95-110 S) ofRaji [RH]DNA recovered from glycerol gradients. The arrow

shows the position of phage PM2 DNA in relation to that of RajiDNA as determined in a separate experiment. *--*, Raji[3H]DNA; A--A, EBV cRNA hybridized.

contained approximately 30 ng of DNA, and 10 ng of EBVDNA.

Properties of 65S Form. The [3H ]DNA from the 65S regionof the glycerol gradients was also analyzed on propidium di-iodide/CsCl gradients in an analogous fashion. In this case,

all the [THIDNA banded at the position of linear DNA.More than 95% of the EBV DNA sequences present bandedtogether with the cellular DNA.To obtain more accurate data on the sedimentation proper-

ties of the 65S form of EBV DNA, the cosedimentation ex-

periments with T4 DNA were repeated for longer times (Fig.5). Under these conditions, the 100S form is pelleted at thebottom of the tube, but the difference in sedimentation ratebetween the 65S form of EBV DNA and T4 DI4A is amplified.In a comparison with the data in Fig. 1, it is clear that theintracellular EBV DNA form sediments more rapidly thanEBV DNA from virus particles.The 65S form could conceivably be due to a linear EBV

DNA molecule, to which proteins or other compounds were

attached. However, when a phenol extraction step was in-cluded in the DNA preparation procedure, most of the EBVDNA sequences present in a high-density DNA fraction fromCsCl gradients still sedimented as a distinct peak at 65 S,as revealed by experiments similar to that shown in Fig. 5.In additional control experiments, it was shown that the 65Sform is also the major EBV DNA form found in growing Rajicells not exposed to [3H]thymidine. In none of these experi-ments was a peak of EBV DNA of 58-59 S detected. We,therefore, conclude that DNA molecules of the type found inEBV particles are not present in Raji cells.

DISCUSSION

It was first proposed by Nonoyama and Pagano (3), on thebasis of alkaline glycerol gradient centrifugation experimentswith Raji cell DNA, that nonintegrated EBV DNA mole-cules are present intracellularly in nonproducer lymphoid celllines. With the same material, Tanaka and N6noyama (4)subsequently demonstrated that EBY DNA molecules withthe apparent properties of those isolated from virus particlescould also be separated from the cellular DNA in neutralglycerol gradients. The present data explicitly confirm the

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FIG. 5. Fractionation of high-density DNA from Raji cellson a neutral glycerol gradient. The experimental conditions were

as in Fig. 3, but the time of centrifugation was extended to 5 hr.*-*, Raji [3H]DNA; 0-- -0, phage T4 [14C]DNA;A --A,

EBV cRNA hybridized.

existence of nonintegrated EBV genomes in Raji cells. How-ever, we disagree with the quoted studies on two points: (a)the nonintegrated form of EBV DNA is not the same structureas that found in virus particles, but appears to be a circularDNA molecule, and (b) all EBV DNA sequences in Raji cellsare not present in a free form.

Plasmids in bacterial systems are circular DNA molecules.It seems, therefore, reasonable that the latent nonintegratedEBV DNA in carrier cells would likewise have a circular DNAconformation, as indicated by the present data. An alternativepossibility is that the 100S and 65S forms represent two typesof replication intermediates or transcription complexes (20),but the relation between the sedimentation coefficients ob-served here makes such an interpretation less likely. Tanakaand Nonoyama (4) have reported that the main intracellularform of EBV DNA in Raji cells has the same sedimentationproperties in neutral solution as the EBV DNA recoveredfrom virus particles. However, as they poured Raji cell lysatesdirectly on top of glycerol gradients, the ratio of viral DNAsequences to cellular DNA in their experiments was muchsmaller than in our studies with prefractionated material.Because of the limited capacity of glycerol gradients for high-molecular-weight DNA, it then became necessary to collectlarge fractions in order to have sufficient material for nucleicacid hybridization and this, in combination with the absenceof an internal size marker, probably precluded the detectionof the relatively small difference in sedimentation rate be-tween the 58-59S form and the 65S form of EBV DNA.We have previously presented evidence that some of the

EBV DNA present in Raji cells is covalently bound in a

linear fashion to host cell DNA (7, 8). While the present data(Fig. 5) indicate a dominance of nonintegrated EBV se-

quences, many of the virus-cell DNA "joint molecules"previously characterized were removed here in the preliminaryfractionation step shown in Fig. 2. It is, therefore, difficult toestimate the exact proportions of nonintegrated versus inte-grated EBV DNA sequences in Raji cells. Due to the natureof the experimental evidence for integration, the numberswill be quite different depending on whether the integratedsequences represent complete EBV genomes, or fragments ofvirus DNA. A reasonable present estimate would be that 5-40% of the EBV DNA in Raji cells is integrated.The 65S form of EBV DNA described here is not unique

to Raji cells. By the same techniques as used in the present

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Epstein-Barr Virus DNA in Carrier Cells 1481

work, 65S EI3V DNA has also been found to be the majorintracellular EBV form recovered from the F265 cell line,an EBV nonproducer lymphoblastoid line established from anormal individual (C. Dierich, A. Adams, T. Lindahl, and G.Klein, manuscript in preparation). Further, in early pre-liminary experiments with the alkaline sucrose gradient pro-cedure of Freifelder et al. (21) covalently closed DNA circleswith the size and density of EBV DNA have sporadicallybeen found in several Burkitt-lymphoma-derived cell lines,but never in a number of EBV-negative control cell lines (T.Lindahl and U. Jehn, unpublished results), but these studieshave so far not been complemented with nucleic acid hybrid-ization experiments.The apparent existence of both nonintegrated circular EBV

gehomes and integrated EBV DNA in Raji cells demonstratesthe complexity of the herpesvirus carrier state. In this regard,large temperate bacteriophages such as P1, which can exist inboth nonintegrated and integrated forms (19, 22), may pro-vide useful experimental models for herpesvirus latency.

We thank Dr. G. Klein for helpful discussions, and Dr. U.Jehn for participating in early attempts to find circular forms ofEBV DNA. This work was supported by grants to both authorsfrom the Swedish Cancer Society and by a grant to T.L. fromthe Swedish Natural Science Research Council. The work wasfurther supported by Contract no. 1 CP33316 within the VirusCancer Program of the U.S. National Cancer Institute, awardedto G. Klein.

1. zur Hausen, H. (1975), "Oncogenic Herpesviruses," inBiochim. Biophys. Acta Rev. on Cancer, in press.

2. Miller, G., Robinson, J., Heston, L. & Lipman, M. (1974)Proc. Nat. Acad. Sci. USA 71, 4006-4010.

3. Nonoyama, M. & Pagano, J. S. (1972) Nature New Biol.238, 169-171.

4. Tanaka, A. & Nonoyama, M. (1974) Proc. Nat. Acad. Sci.USA 71, 4658-4661.

5. Pulvertaft, R. J. V. (1965) J. Clin. Pathol. 18, 261-273.6. Nonoyama, M. & Pagano, J. S. (1973) Nature 242, 44-47.7. Adams, A., Lindahl, T. & Klein, G. (1973) Proc. Nat. Acad.

Sci. USA 70, 2888-2892.8. Adams, A. & Lindahl, T. (1974) in Herpesviruses and

Oncogenesis, eds. zur Hausen, H., Epstein, M. A. & de The,G. (Int. Agency Res. Cancer, Lyon), in press.

9. Yagil, E. & Rosner, A. (1970) J. Bacteriol. 103, 417-421.10. Hudson, B., Upholt, W. B., Devinny, J. & Vinograd, J.

(1969) Proc. Nat. Acad. Sci. USA 62, 813-820.11. Lindahl, T., Klein, G., Reedman, B. M., Johansson, B. &

Singh, S. (1974) Int. J. Cancer 13, 764-772.12. Klein, G., Gergely, L. & Goldstein, G. (1971) Clin. Exp.

Immunol. 8, 593-602.13. Hinuma, Y., Konn, M., Yamaguchi, J., Wudarski, D. J.,

Blakeslee, J. R., Jr. & Grace, J. T., Jr. (1967) J. Virol. 1,1045-1051.

14. Jehn, U., Lindahl, T. & Klein, G. (1972) J. Gen. Virol. 16,409-412.

15. Freifelder, D. (1970) J. Mol. Biol. 54, 567-577.16. Gergely, L., Klein, G. & Ernberg, I. (1971) Virology 45,

10-21.17. Tomizawa, J. I. & Anraku, N. (1964) J. Mol. Biol. 8, 516-

540.18. Helinski, D. R. & Clewell, D. B. (1971) Ann. Rev. Biochem.

40, 899-942.19. Ikeda, H. & Tomizawa, J. I. (1968) Cold Spring Harbor

Symp. Quant. Biol. 33, 791-798.20. Doerfler, W., Lundholm, U., Rensing, U., & Philipson, L.

(1973) J. Virol. 12, 793-807.21. Freifelder, D., Folkmanis, A. & Kirschner, I. (1971) J.

Bacteriol. 105, 722-727.22. Scott, J. (1970) Virology 40, 144-151.

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