Quantitation of Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) by a two-site enzyme immunoassay, in parallel with EBV-DNA

Journal of Immunological Methods, 89 (1986) 151-158 Elsevier

151

JIM 03889

Quantitation of Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) by a two-site enzyme immunoassay, in parallel with EBV-DNA Lars Stern~s, L e n a Eliasson, R i c h a r d L e r n e r and G e o r g e Klein Department of Tumor Biology, Stockholm, Sweden, and Research Institute of Scripps Clinic, La Jolla, CA 92037, U.S.A. Received 22 November 1985, accepted 11 December 1985)

A two-site enzyme (TSE) lmmunoassay was developed for the quantitatlon of the Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) using a rabbit serum raised against a synthetic peptide derived from the BamHI K region of the viral genome. Comparison of 12 EBNA-positive and 3 negative cell lines proved that the test was EBV-specific. A dot-blot assay utilizing cloned and nick translated EBV-DNA BamHI M fragment confirmed the EBV-carrier status of the EBNA-positive lines. The results obtained with both the TSE immunoassay and dot-blot assay were in agreement with published values. In contrast to earlier reports, we could not demonstrate any correlation between the content of EBNA and the number of viral genome copies. Key words: Epstein-Barr virus (EB V); Epstein-Barr virus-determined nuclear antigen (EBNA): EB V-DNA; ELISA for EBNA; Two-site enzyme immunoassay

Introduction

The Epstein-Barr virus-determined nuclear antigen (EBNA) can be regularly detected by the ACIF reaction (Reedman and Klein, 1973) in virally immortalized B-lymphocytes and in Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC) biopsies (Reedman and Klein, 1973; Klein et al., 1974a). It is the first and often the only detectable viral antigen that appears in EBVinfected B-lymphocytes, before the onset of cellular DNA synthesis (Einhorn and Ernberg, 1978). EBV-carrying lymphoid lines and tumors usually contain multiple copies of the viral genome (Zur Hausen et al., 1972). Most of them are carried as free episomes in the cell nucleus (Nonoyama and Pagano, 1972; Lindahl et al., 1976).Transformed cells replicate viral DNA concomitantly with the cellular DNA (Hampar et al., 1974; Thorley-Lawson and Strominger, 1976).

Previous comparisons of the amount of EBNA and the number of EBV genomes per cell, performed on a limited number of cell lines and derived hybrids have suggested that a direct correlation may exist between the two parameters (Ernberg et al., 1977, 1983; Shapiro et al., 1979). This suggests that the expression of EBNA is an autonomous function of the latent viral genomes. EBV-positive cell lines, or EBV-negative lines transfected with the B a m H I K fragment of the viral DNA, known to encode the EBNA-1 antigen (Summers et al., 1982; Fischer et al., 1984; Hearing et al., 1984) can sustain the replication of plasmids that contain the EBNA-1 binding ori p region of the viral genome indicating that the maintenance of the viral genomes in the episomal form may be a function of EBNA-1 (Yates et al., 1984) The size of the EBNA-1 antigen varies between different virus strains. This variation has been

0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

152 shown to be related to differences in the size of the third internal repeat (IR 3) region of the viral genome contained within the BamH! K fragment (Hennesy et al., 1983). Antiserum raised against a bacterial fusion protein that contained part of the IR 3 repeat (Hennesy and Kieff, 1983) and against a synthetic peptide derived from the nucleotide sequence within the IR 3 repeat (Dillner et al., 1984) confirmed that this region codes for an EBV-specific nuclear protein as defined by the A C I F reaction and by Western blotting. A second virally coded nuclear antigen has recently been detected by transfecting EBV negative cells with subgenomic viral fragments corresponding to the BamHI W Y H region and by raising antibodies against bacterial fusion proteins containing sequences from these regions or synthetic peptides deduced from the nucleotide sequence within them (Rymo and Klein, 1985; Hennesy and Kieff, 1985; Dillner et al., 1985b). This antigen has been designated EBNA-2. Cells with a deleted EBNA-2 region, for example P3HR-I and Daudi absorb EBNA-1 but not EBNA-2 antibodies. Such absorbed sera can stain lines that contain both EBNA-1 and EBNA-2, but not the relatively frequent EBNA-2-negative Burkitt lymphoma lines (Dillner et al., 1985a). Previously, we have developed a monospecific reagent against the B a m H I K encoded EBNA-1 antigen, by immunizing rabbits with a synthetic Gly-Ala peptide, deduced from the IR 3 sequence (Dillner et al., 1984). This reagent was used to develop a two-site enzyme immunoassay to quantitate EBNA-1. Using this method, we have re-examined the relationship between EBNA-1 and the number of EBV genomes carried by different growth transformed cells. Materials and methods

Preparation of cell extracts The cells were harvested, pelleted and frozen at - 2 0 ° C , in aliquots. They were thawed by adding 1 ml of 20 m M Tris-HCl pH 7.5, 150 mM NaC1, 1 mM EDTA and 1 mM PMSF, resuspended and centrifuged at 4°C for 10 min in an Eppendorff centrifuge. The supernatant was collected and the protein concentration determined by the method of Bradford (1976).

Two-site enzyme immunoassav Antipeptide serum was affinity purified on the corresponding peptide attached to AH-Sepharose 4B (Pharmacia) as described in the manufacturer's manual. The immunoaffinity-purified immunoglobulin was diluted in 50 mM bicarbonate buffer pH 9.6 and coated to microtiter plates (Flow, M 129 A) by incubation o.n. at room temperature. After washing three times with 20 mM Tris-HCl pH 7.5, 150 mM NaCl with 0.05% Tween 20 (TNT), the plates were incubated at 4°C with serial 2-fold dilutions of cell extracts diluted in T N T with 2% ovalbumin ( T N T / o v a ) for I h. After washing five times with TNT, an antiEBNA-positive human serum diluted in T N T / o v a was added and incubated for 1 h, at room temperature. After five times washing with T N T / o v a anti-human IgG conjugated to alkaline phosphatase was added (Sigma, diluted 1:1000 in T N T / o v a ) together with normal rabbit serum and incubated for 1 h at room temperature. After washing five times with TNT. the substrate was added, consisting of 1 mg of o-nitrophenyl phosphate (Sigma) per ml ot a 0.1 M Tris-glycine pH 10.4, 1 mM MgC1. 1 mM ZnCI solution. The absorbance at 405 nm was determined after allowing the enzyme reaction to proceed for 60 rain at room temperature. All tests were performed in duplicate. The Raji cell line was always included as a positive control. The pellet remaining after extraction was further extracted with 2 M NaC1 and 4 M urea. However, this did not lead to the extraction of more antigen, suggesting that most of the antigen was already extracted. Preparation of DNA D N A was prepared according to the method of Blin and Stafford (1976). Briefly, the cells were washed, pelleted and resuspended in 0.5 M EDTA, 0.5% SDS and 100 # g / m l proteinase K (Sigma). After incubation for 3 h at 50°C, the D N A was extracted three times with phenol and twice with chloroform : isoamyl alcohol (24 : 1). The samples were dialyzed and treated with RNase (Sigma). After an additional extraction with phenol and chloroform:isoamyl alcohol, the D N A was dialyzed extensively against TE buffer (10 mM Tris p H 8.0, 1 mM EDTA).

153

Dot-blot assay for quantitation of EBV genomes Extracted D N A was dotted onto nitrocellulose filters according to the method of Andre (Andre, 1979). 3-5 fig D N A were dissolved in 170 t~l Tris-Hcl p H 7.4, 30 /~1 2 N N a O H , 100 ffl 20 x SSC. The D N A was heated to 80°C for 10 min and subsequently neutralized with 40/~1 2 M TrisHC1. The D N A was dotted onto a nitrocellulose membrane filter using a Schleicher and Schuell 96-well microsample filtration manifold. The filter was left at room temperature o.n. and then baked for 2 h at 80°C. The D N A from each cell line was dotted onto the filter in a 2-fold dilution series of at least four steps, starting with 5 fig DNA. Calf thymus D N A (2.5 fig) was added to dilutions with less than 2.5 /~g DNA. Tests were performed in duplicate. As a standard, the BamHI M restriction fragment cloned into p B R 322 (Arrand et al., 1981), was dotted onto each filter in a 2-fold dilution series corresponding to a range from 50 down to 0.37 EBV genomes per cell. 2.5 fig calf thymus D N A was added as a carrier to each dilution step. Calf thymus D N A was dotted onto the filters as a negative control.

Results

Quantitation o / B a m H I K EBNA Representative titration curves for four of the tested cell lines are shown in Fig. 1. The EBNA content of different lines, as measured by the two-site enzyme ( T S E ) i m m u n o a s s a y , was expressed in relative units as the difference in the number of 2-fold antigen dilution steps required to obtain an OD value of 0.6, compared to Raji included as the standard positive control. Table I shows these relative logarithmic values after conversion to linear values relative to the Raji line which was assigned the arbitrary valt~e of 100%. The EBV genome and EBNA-negative Ramos, B JAB and Lukes lines were negative, whereas the EBV-carrying, non-producing BL lines Rael, Daudi, Namalwa and the lymphoblastoid cell line

2.0-

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o

o

o

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Filter hybridization The filter was prehybridized in 5 X Denhard's solution and 1 x SSC for 3 - 6 h at 37°C. The hybridization was carried out for 24-48 h at 42°C in 50% formamide, 6 x SSC, 10 m M EDTA, 0.5% SDS and 100 /~g/ml sheared calf thymus DNA. To this mixture the nick-translated (P 32-labelled) and boiled BamHI M plasmid (Andre, 1979) was added. After incubation the filter was washed free of excess plasmid by washing three times, for 5 min, in 2 x SSC, 0.1% SDS at room temperature, followed by two washings, for 30 min at 65°C in 0.1 × SSC and 0.1% SDS.

o

o

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A utoradiography The filter was autoradiographed with Fuji X-ray film, between intensifying screens, at - 7 0 ° C .

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500

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Scintillation counting After autoradiography, each dot was counted in a Rack Beta (LKB) liquid scintillation counter.

Protein Fig.

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Bjab/B95-8, 5pg DNA Fig. 2. Dot-blot for EBV-DNA. The cell lines Raji, 6410, BJAB and BJAB/B95-8 were compared to an EBV genome standard. A P32 nick translated plasmid containing the viral BamHI M fragment was used as the hybridization probe. Row A: The BamHl M plasmid applied in concentrations equivalent to a range from 50 to 0.37 EBV genome copies per cell. Rows B through G are sample DNA. All samples were applied in 2-fold dilution series starting with 5 #g DNA. Row B and C, lanes 1-8: Raji. Row D and E, lanes 1-8: 6410. Row F and G, lanes 1-4: BJAB. Row F and G, lanes 5-8: BJAB/B95-8.

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Fig. 3. BamH1K EBNA content in 15 cell lines, as measured by the TSE immunoassay, plotted against the viral genome copy numbers, as measured by the dot-blot assay. The EBNA content is expressed relative to the Raji line.

155 TABLE I CELL LINES TESTED IN THE TWO-SITE ENZYME IMMUNOASSAY AND THE DOT-BLOT ASSAY FOR EBV-DNA Name

Ref.

EBV-carrier status a

EBV genomesTcell b

EBNA-1 content c

Lukes BJAB BJAB/B95-8 Ramos AW Ramos Ramos HRIK Ramos B95-8 EHR-D Ramos EHR-H Ramos EHR-O Ramos Namalwa 6410 Rael Raji Daudi

1 2 3 4 4 3 5 d d d 6 7 8 9,10 11

neg neg pos neg pos pos pos pos pos pos pos pos pos pos pos

0 0 3.0 _+0.5 0 0.7 ± 0.2 1.5 ± 0.5 1.3 _+0.5 1.4 ± 0.5 1.3 ± 0.4 0.8 ± 0.1 1.0 ± 0.4 40.0 ± 3.0 88.0 ± 1.1 64.0 ± 4.7 111.0 ± 5.0

0 0 35.7 ± 0 3.1 ± 12.5 ± 2.2 ± 1.6 ± 2.2 ± 3.1 ± 35.7 ± 25.0 ± 12.5 ± 100.0 6.3 ±

10.7 0.9 3.8 0.7 0.5 0.7 0.9 10.7 7.5 3.8 1.9

a According to presence of EBNA as determined by ACIF and presence of viral genomes. b EBV genome copies per cell were determined by the dot-blot assay. The results are the mean of two determinations, each performed in duplicate. SD is indicated. c EBNA-1 values were measured by the TSE immunoassay and are expressed relative to the Raji cell line which was included in all tests as an internal standard. It was assigned the arbitrary value of 100%. The results are the mean of two determinations, each performed in duplicate. SD is indicated. d G. Klein and A. Westman, unpublished. References: 1, Van Santen et al., 1981; 2, Klein et al., 1974b; 3, Fresen and Zur Hausen, 1976; 4, Klein et al., 1975; 5, Klein et al., 1976; 6, Klein et al., 1972b~ 7, Ikawata and Grace, 1964; 8, Klein et al., 1972a; 9, Pulvertaft, 1964; 10, Epstein et al., 1966; 11, Klein et al., 1968.

( L C L ) 6410 w e r e all p o s i t i v e in the T S E i m m u n o assay. E B V - n e g a t i v e B L s c o n v e r t e d w i t h the B95-8 v i r a l s u b s t r a i n (B J A B / B 9 5 - 8 ) o r the P 3 H R - 1 substrain (AW-Ramos, EHR-O-Ramos, EHR-DR a m o s , E H R - H - R a m o s ) w e r e also positive. T h e r e l a t i v e a m o u n t s o f E B N A m e a s u r e d b y the T S E i m m u n o a s s a y w e r e in a g r e e m e n t w i t h t h e p r e v i o u s d a t a of E r n b e r g et al. as far as the lines R a e l , A W - R a m o s , B J A B / B 9 5 - 8 a n d 6410 are c o n c e r n e d ( E r n b e r g et al., 1977; E r n b e r g et al., 1983). T h e N a m a l w a l i n e c o n t a i n e d a p p r o x i m a t e l y t w i c e the a m o u n t of E B N A in o u r assay c o m p a r e d to the v a l u e o b t a i n e d b y E r n b e r g et al., w h o u s e d a quantitative microfluorimetric method. The EBV g e n o m e c o p y n u m b e r p e r cell was f o u n d to b e 3, in c o n t r a s t to o u r o b s e r v e d v a l u e of 1. T h e R a m o s / B 9 5 - 8 line c o n t a i n e d a p p r o x i m a t e l y ten t i m e s less E B N A t h a n r e p o r t e d in the e x p e r i m e n t s of E r n b e r g et al. T h e r e p o r t e d E B V - D N A v a l u e for this cell lines was in g o o d a g r e e m e n t w i t h o u r

result, 1 v e r s u s 1.3 g e n o m e c o p i e s p e r cell. If the E B N A - p o s i t i v e h u m a n s e r u m was r e p l a c e d b y an E B V - n e g a t i v e s e r u m , the T S E i m m u n o a s s a y was n e g a t i v e w i t h all cells. T h e s a m e was t r u e if the a n t i - G l y - A l a p e p t i d e a n t i s e r u m was s u b s t i t u t e d w i t h a r a b b i t a n t i b o d y d i r e c t e d against an irrelev a n t p e p t i d e . M o r e o v e r , the G l y - A l a p e p t i d e b l o c k e d the T S E i m m u n o a s s a y , w h e r e a s an irrelev a n t p e p t i d e d i d n o t interfere. A s r e p o r t e d e l s e w h e r e ( H a m m a r s k j t ~ l d et al., 1985) C V 1 cells t r a n s f e c t e d w i t h a BamHI K f r a g m e n t c a r r y i n g e x p r e s s i o n v e c t o r w e r e also rea c t i v e in o u r T S E i m m u n o a s s a y for E B N A , a n d in the A C I F assay, w h e r e a s cells t r a n s f e c t e d w i t h the s a m e v e c t o r w i t h o u t the viral i n s e r t w e r e n e g a t i v e .

Quantitation of EBV-DNA Q u a n t i t a t i o n of viral D N A was p e r f o r m e d b y a d o t - b l o t assay u s i n g an E B V BamHI M p r o b e . T h i s f r a g m e n t w a s c h o s e n b e c a u s e it r e p r e s e n t s a n o n - r e p e t i t i v e r e g i o n of the viral g e n o m e a n d was

156 not deleted in any cell line so far tested. The probe did not hybridize to EBV-negative human D N A or to calf thymus carrier DNA. In order to use it as a standard for EBV genome quantitation, the EBV B a m H I M plasmid was dotted onto the nitrocellulose filter in a 2-fold dilution series corresponding to EBV genome copy numbers between 50 and 0.37 per cell. The number of EBV genome equivalents per cell was calculated from the number of base pairs in the EBV genome (173 kb), the B a m H I M fragment (4.7 kb) and the human genome (3 × 109 base pairs). In order to get a linear relationship between the dilution factor and the hybridization intensities, the amount of D N A dotted onto the filter had to be between 2 and 4 /~g. Therefore 2.5 /~g carrier D N A was added to all samples containing 1.25/~g or less. The number of EBV genome copies was estimated by autoradiography and by liquid scintillation counting of the individual dots. The cpm for the standard dots were plotted against the EBV genome copy number. The results are included in Table I. The EBNA-negative lines Lukes, B JAB and Ramos were EBV-DNA negative as expected. The EBV-converted Ramos and B JAB lines and the EBV-carrying BL line Namalwa contained less than 5 EBV genomes per cell. Four of the tested lines Raji, 6410, Rael and Daudi contained between 40 and 110 EBV genome copies per cell. With the exception of the 6410 line which was found to contain 40 genomes, in contrast to the earlier published value of 20, and Namalwa which we found to contain 3 genomes per cellin contrast to the result reported by Ernberg et al. of one copy, all the tested cell lines contained EBV genomes to the same level as in earlier papers (Ernberg et al., 1977, 1983; Matsuo et al., 1984). In Fig. 3, the relative amounts of EBNA and E B V - D N A have been plotted against each other. There is no obvious correlation, except for the fact that the cell lines with 40-110 EBV genomes per cell have a relatively high content of EBNA-1 and EBV-DNA-negative lines are EBNA negative.

Discussion We have established a TSE immunoassay for EBNA-1 based on a complementary pair of antibodies: a rabbit antibody directed against a GlyAla repeat (IR 3) within the EBNA-l-encoding B a m H I K region and an EBV antibody-positive h u m a n serum. Eleven EBNA-positive BE lines, including several independently EBV-converted sublines of the originally EBV-negative Ramos and B JAB lymphomas and one LCL, 6410, were all positive in the TSE immunoassay, whereas 3 EBV-negative lymphoma lines were negative. The dot-blot assay for viral DNA, performed simultaneously, confirmed the EBV status of the lines. The EBV specificity of the immunoassay was further confirmed by the fact that substitution of the EBV-positive human serum with a negative serum or exchange of the anti-Gly-Ala peptide serum for a rabbit antiserum directed against an irrelevant peptide abolished the reaction. Moreover, the reaction was inhibited by the addition of the free Gly-Ala peptide, whereas an irrelevant peptide had no effect. The specificity of the test was also c o n f i r m e d by the finding of HammarskjiSld et al. (1985) that CV-1 cells transfected with a B a m H I K carrying expression vector gave a positive reaction whereas controls transfected with the same vector without the EBV insert were negative. Previously, Ernberg et al. (1977) measured the amount of EBNA in various EBV-carrying lines by cytofluorimetry. They found a correlation between the amount of EBNA and the number of EBV genome copies. Our study shows no significant correlation apart from the fact that the 4 cell lines with the highest EBV copy number (40-110) also had the highest quantities of EBNA-1 and EBV-DNA-negative lines were also EBNA-1 negative. The lack of a more precise correlation in our admittedly limited material also advocates caution against generalizing from studies on a small number of lines. In the study by Ernberg et al. (1977), the situation was further complicated by the fact that several of the lines were actually somatic hybrids between two of the standard lines, Raji and Namalwa. They have contributed strongly to the

157 correlation that was observed, b u t the significance of this f i n d i n g is d i m i n i s h e d b y the fact that they c a n n o t be regarded as i n d e p e n d e n t lines. In a comparison between near-diploid and n e a r - t e t r a p l o i d Raji s u b l i n e s a n d R a f t / R a f t somatic hybrids, Shapiro et al. (1979) also f o u n d a linear correlation b e t w e e n the average n u m b e r of EBV g e n o m e copies per cell a n d the relative a m o u n t of E B N A , measured b y c o m p l e m e n t fixation. However, these lines are closely interrelated a n d the study is therefore not c o m p a r a b l e to our m e a s u r e m e n t o n i n d e p e n d e n t or i n d e p e n d e n t l y converted lines. A n o t h e r reason for the discrepancies b e t w e e n earlier results a n d our present findings m a y be sought in the fact that the A C I F reaction detects b o t h EBNA-1 a n d E B N A - 2 while our test is specific for EBNA-1. A n a d d i t i o n a l c o m p l i c a t i o n m a y stem from the recent detection of two add i t i o n a l n u c l e a r antigens, E B N A - 3 a n d -4 by W e s t e r n b l o t t i n g ( K a l l i n et al., 1985). A possible f u n c t i o n a l role of EBNA-1 is suggested by its ability to b i n d to one origin of replication (ori p) o n the EBV g e n o m e a n d thereby c o n t r i b u t e to the m a i n t e n a n c e of EBV in the free plasmid form (Yates et al., 1984). T h e high EBNA-1 expression of the N a m a l w a line is interesting in this respect. N a m a l w a is a n exceptional line, k n o w n to carry one g e n o m e copy per cell in a n integrated form a n d n o free EBV copies ( H e n d e r s o n et al., 1983; M a t s u o et al., 1984). The high a m o u n t s of EBNA-1 expressed in this line m a y reflect the d y s f u n c t i o n of the mechan i s m that n o r m a l l y regulates the b a l a n c e between EBNA-1 a n d the episomal viral genomes.

Acknowledgement This work was s u p p o r t e d by N I H G r a n t 5 RO1 CA28380-05, Swedish C a n c e r Society a n d Sysk o n e n Svenssons F u n d .

References Andre, J., 1979, Schleicher and Schuell, Sequences Application Update, no. 363.

Arrand, J., L. Rymo, J. Walsh, E. BjOrck, T. Lindahl and B. Griffin, 1981,~Nucl. Acids Res. 9, 2999. Blin, N. and D.W. Stafford, 1976, Nucl. Acids. Res. 3, 2303. Bradford, M.M., 1976, Anal. Biochem. 72, 248. Dillner, J., L. Sternks, B. Kallin, H. Alexander, B. EhlinHenriksson, H. Jtrnvall, G. Klein and R. Lerner, 1984, Proc. Natl. Acad. Sci. U.S.A. 81, 4652. Dillner, J., B. Kallin, B. Ehlin-Henriksson, L. Timar and G. Klein, 1985a, Int. J. Cancer 35, 359. Dillner, J., B. Kallin, G. Klein, H. JiSrnvall, H. Alexander and R. Lerner, 1985b EMBO J. 4, 1813. Einhorn, U and Ernberg, 1978, Int. J. Cancer 21,157. Epstein, M.A., B.G. Achong, Y.M. Barr, B. Zajac, G. Henle and W. Henle, 1966, J. Natl. Cancer Inst. 37, 547. Ernberg, I., M. Andersson-Anvret, G. Klein, G. Lundin and D. Killander, 1977, Nature (London) 266, 269. Ernberg, I., G. Klein, B.C. Giovanella, J. Stehlin, K.J. McCormick, M. Andersson-Anvret,P. Aman and D. Killander, 1983, Int. J. Cancer 31, 163. Fischer, D.K., M.F. Robert, D. Shedd, W.P. Summers, J.E. Robinson, J. Wolak, J.E. Stefano and G. Miller, 1984, Proc. Natl. Acad. Sci. U.S.A. 81, 43. Fresen, K.O. and H. Zur Hausen, 1976, Int. J. Cancer 17, 161. HammarskjiSld, M.L:, J. Dillner, S.S. Wang and G. Klein, 1985, submitted. Hampar, B., A. Tanaka, M. Nonoyama and J. Derge, 1974, Proc. Natl. Acad. Sci. U.S.A. 71,631. Hearing, J.C., J.C. Nicolas and A.J. Levine, 1984, Proc. Natl. Acad. Sci. U.S.A. 81, 4373. Henderson, A., S. Ripley, M. Heller and E. Kieff, 1983, Proc. Natl. Acad. Sci. U.S.A. 80, 1987. Hennesy, K. and E. Kieff, 1983, Proc. Natl. Acad. Sci. U.S.A. 80, 5665. Hennesy, K. and E. Kieff, 1985, Science 277, 1238. Hennesy, K., M. Heller, V. Van Santen and E. Kieff, 1983, Science 220, 1396. Ikawata, S. and J. Grace, 1964, N.Y. State J. Med. 64, 2279. Kallin, B., J. Dillner, I. Ernberg, B. Ehlin-Henriksson, A. Rosen and G. Klein, 1985, submitted. Klein, E., G. Klein, J. Nadkarni, H. Wigzell and P. Clifford, 1968, Cancer Res. 28, 1300. Klein, G., L. Dombos and B. Gothoskar, 1972a, Int. J. Cancer 10, 44. Klein, G., R. Van Furth, B. Johansson, I. Ernberg and P. Clifford, 1972b, Cambridge Symposium on Oncogenesis and Herpesviruses, eds. Biggs, deThe and Payne, p. 253. Klein, G., B.C. Giovanella, T. Lindahl, P. Fialkow, S. Singh and J. Stehlin, 1974a, Proc. Natl. Acad. Sci. U.S.A. 71, 4737. Klein, G., T. Lindahl, M. Jondal, W. Leibold, J. Menezes, K. Nilsson and C. Sundstrt~m, 1974b, Proc. Natl. Acad. Sci. U.S.A. 71, 3283. Klein, G., B. Giovanella, A. Westman, J. Stehlin and I9. Mumford, 1975, lntervirology 5, 319. Klein, G., J. Zeuthen, P. Terasaki, R. Billing, R. Honig, M. Jondal, A. Westman and G. Clements, 1976, Int. J. Cancer 18, 639.

158 Lindahl, T., A. Adams, G. Bjursell, G.W. Bornkamm, C. Kaschka-Dierich and U. Jehn, 1976, J. Mol. Biol. 102, 511. Matsuo, T., M. Heller, L. Petti, E. Oshiro and E. Kieff, 1984, Science 226, 1322. Nonoyama, M. and J. Pagano, 1972, Nature (London) 238, 169. Pulvertaft, R.J.V., 1964, Lancet i, 238. Reedman, B.M. and G. Klein, 1973, Int. J. Cancer 11,499. Rymo, L. and G. Klein, 1985, Proc. Natl. Acad. Sci. U.S.A. 82, 3435.

Shapiro, I., J. Luka, M. Andersson-Anvret and G. Klein, 1979, lntervirology 12, 19. Summers, W.P., E.A. Grogan, D. Shedd, M. Robert, C.R. Liu and G. Miller, 1982, Proc. Natl. Acad. Sci. U.S.A. 79, 5688. Van Santen, V., E. Cheung and E. Kieff, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 1930. Yates, J., N. Warren, D. Reisman and B. Sugden, 1984, Proc. Natl. Acad. Sci. U.S.A. 81, 3806. Zur Hausen, H., V. Diehl, H. Wolf, H. Schulte-Holthausen and U. Schneider, 1972, Nature (London) 237, 189.