CharacteriZation of Human B-Cell Lines Harbouring ...

J. gen. Virol. (1984), 65, 1781-I789.

Printed in Great Britain

1781

Key words: human retrovirus/EB virus~B-cells

CharacteriZation of Human B-Cell Lines Harbouring Both Adult T-Cell LeukaemiaL(ATL) Virus and Epstein-Barr Virus Derived from ATL Patients x

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By Y O S H I O K O Y A N A G I , N A O K I Y A M A M O T O , * N O B U Y U K I KOBAYASHI, KANJ! HIRAI, t HIROSHI KONISHI, KAORU TAKEUCHI, YUETSU TANAKA,t MASAKAZU HATANAKAAND YORIO HINUMA

Institute for Virus Research, Kyoto University, Shogoin, Kyoto 606, Japan and I Department of Molecular Biology, Tokai University School of Medicine, Bohseidai, Isehara 259-11, Japan (Accepted 4 June 1984) SUMMARY

Human B-cell lines, designated ATLB cell lines, were spontaneously established from peripheral blood of adult T-cell leukaemia (ATL) patients, The cell lines consistently expressed ATL-associated antigen (ATLA) and Epstein Barr virusassociated nuclear antigen (EBNA). A cloned ATLB line, designated ATLB 2, showed that both ATLA and EBNA antigens were present in the same B-cell clone. In this study, we have further characterized ATLV and EBV in the cloned ATLB 2 cell line by biochemical techniques. The ATLA antigens in these clones, initially shown by immunofluorescence, were studied by immunoprecipitation with human sera from ATL patients and the Western blotting technique using a mouse monoclonal antibody (GIN-14). We identified ATLV core polypeptides 24K and 19K in the ATLB cell extracts. Furthermore, total cellular D N A and poly(A) RNA from the ATLB cell lines were analysed for the presence of viral genomes with molecularly cloned D N A probes containing the ATLV and EBV sequence, respectively. The results showed that all ATLB 2 clones contained highly conserved ATLV proviral D N A irrespective of whether or not they expressed ATLA. They also contained several copies of EB virus D N A and D N A - D N A reassociation kinetics studies clearly showed that most of the EBV DNA sequences were present in ATLB cells. ATLV-related m R N A was detected in only ATLA-positive clones (ATLB 2-3 and 2-21) but not in a negative clone (ATLB 2-45). INTRODUCTION

Both herpesviruses and retroviruses are widely distributed in the animal kingdom and cause various malignancies in different species. Each virus group alone can induce tumours but several observations suggest a collaborative role between these two virus groups in the pathogenesis of certain tumours (Deinhardt & Deinhardt, 1979). In man, Epstein-Barr virus (EBV), a herpesvirus, and recently described retroviruses, human T-cell leukaemia virus (HTLV) or adult T-cell leukaemia virus (ATLV), may together play a role in certain tumours (Yamamoto et al., 1982). EBV is known to cause infectious mononucleosis and has been associated with two human malignant tumours, Burkitt's lymphoma and nasopharyngeat carcinoma (NPC) (Henle & Henle, 1979). On the other hand, Poiesz et al. (1980, 1981) first reported the isolation of a typeC retrovirus from patients with cutaneous T-cell lymphoma in the U.S.A., that is termed HTLV (Poiesz et at., 1980, 1981; Posner et al., 1981). Independently, Hinuma and his co-workers isolated a retrovirus from a cell line from patients with ATL (Hinuma et al., 1981 ; Miyoshi et al., 1981 ; Yoshida et al., 1982), a unique leukaemia which is endemic in southwest Japan (Takatsuki t Present address: Department of Immunology, School of Hygienic Science, Kitasato University, Kitasato, Sagamihara 228, Japan. 0022-1317/84/0000-6t 1t $02.00 © 1984 SGM

1782

Y. KOYANAGI AND OTHERS

et al., 1977 ; U c h i y a m a et al., 1977). E x t e n s i v e analysis w i t h s e r o e p i d e m i o l o g i c a l and virological m e t h o d s s h o w e d that H T L V / A T L V is a closely related, if not causative, a g e n t o f A T L ( H i n u m a et al., 1981, 1982; K a l y a n a r a m a n et al., 1982; R o b e r t - G u r o f f et al., 1982). W e recently established continuous cell lines c o m p o s e d o f B-type lymphocytes, d e s i g n a t e d A T L B cell lines, f r o m the p e r i p h e r a l blood l y m p h o c y t e s (PBL) o f several A T L patients. I m m u n o f l u o r e s c e n t studies revealed that m o s t o f t h e m w e r e positive for A T L - a s s o c i a t e d antigen ( A T L A ) and E B V - a s s o c i a t e d nuclear a n t i g e n ( E B N A ) ( Y a m a m o t o et al., 1982). T h e following represents a c o n t i n u a t i o n o f our studies on the possible role o f A T L V - i n f e c t e d B-cells in the genesis o f A T L . Several A T L B cell lines and their subcIones w e r e analysed by b i o c h e m i c a l techniques. T h e d a t a revealed that A T L B cells c o n t a i n e d b o t h A T L V - and EBV-specific D N A s . T h e s e d a t a clearly s h o w t h a t A T L V is c a p a b l e o f infecting and replicating not only in T-cells but also in B-cells. W h e t h e r A T L V contributes to B-cell t r a n s f o r m a t i o n or is a p a s s e n g e r virus in E B V - t r a n s f o r m e d cells r e m a i n s to be d e t e r m i n e d . METHODS Cells. ATLB 2 cells were established from an ATL patient and were cloned by using seaplaque agarose as previously described (Yamamoto et al., 1982). Four cloned B-cell lines, ATLB 2-3, 2-21, 2-23 and 2-45, were used. The B-cell origin of these clones was evidenced by the following observations. Double staining of the fixed cells with anti-human Ig-conjugated rhodamine and anti-mouse lgG-conjugated fluorescein isothiocyanate after applying ATLV-specific monoclonal antibodies clearly demonstrated the co-existence of ATLA and human Igs on the same cells. These cell lines were maintained in RPMI 1640 medium supplemented with 10~ foetal calf serum (FCS), penicillin (100 IU/ml) and streptomycin (100 ~g/ml). They were subcultured routinely every 4 days. Antisera. Sera from an ATL patient and a healthy adult were examined for their reactivity with ATLA by the indirect immunofluorescence test. The serum titres used for this study were 320 from an ATL patient and < 10 from a healthy adult. Monoclonal antibody (GIN-14) raised against p28 and p19 of ATLA was obtained using mouse myeloma cells, as described elsewhere (Tanaka et al., 1983), Radioactive labelling oJ]orotein and immunoprecipitation. Typically, 8 × 106 cells were labelled for 16 h in 10 ml of medium containing 50 ~tCi/ml L-[3SS]methionine (1320 Ci/mmol) (Amersham). To increase incorporation of radioactive precursors, dialysed FCS was used and unlabelled methionine was reduced in labelling experiments to 10% of its concentration in normal medium. Then the cell suspension was washed thoroughly three times with phosphate-buffered saline (PBS) and lysed by adding 0.5 ml of cold low-salt extraction buffer (0.14 M-NaCl, 3 mMMgCI.,, 1 mM-dithiothreitol, 2 mM-phenylmethylsulphonyl fluoride, l0 mM-Tris-HC1 pH 8.0) containing 0.5~ NP40. An NP40 soluble lysate was obtained after vortex mixing and incubation for 20 min on ice, by centrifugation at 12000 g for 10 min at 4 °C. Five vtl of patient's serum or control serum was incubated for 15 h at 4 °C with lysates from cells (50 ~tl). Immunoprecipitates were bound to 10 mg Protein A-Sepharose (Pharmacia) and washed twice in 1 ml high-salt washing buffer (20 mM-Tris-HC1 pH 7.6, 0.5 M-NaC1, 1 mM-EDTA, 0.5% NP40, 1% desoxycholate) containing 0.1% bovine serum albumin (BSA) and twice in the same washing buffer not containing 0.1% BSA. Finally, they were washed twice with low-salt washing buffer (10 mM-Tris-HCl pH 7-6, 10 mM-NaCI). Washed immunoprecipitates were extracted for 3 min at 100 °C in 20 ~tlof sample buffer (0.0625 MTris-HC1 pH 6-8, 2% SDS, 4 ~ 2-mercaptoethanol). After addition of 10H glycerol and 0.05% bromophenol blue, samples were separated on SDS-polyacrylamide gels (Yamamoto & Hinuma, 1982; Yamamoto et al., 1983). Polyaerytamide gel electrophoresis (PAGE). Electrophoresis in SDS-polyacrylamide slab gels was performed in a 12-5% separating gel under reducing conditions with a 5% stacking gel using the discontinuous buffer system of Laemmli (1970). The gels were prepared for fluorography as described by Bonner & Laskey (1974) before drying to visualize radioactive bands using Kodak X-Omat RP film. ~¢Lestern blotting. Proteins in extracts of 1 x 106 ATLB 2-3 and 2-21 cells were separated on 12-5% polyacrylamide gel in the presence of SDS. The resultant protein bands were electrophoretically transferred to nitrocellulose as described by Towbin et al. (1979). Transferred proteins were detected either by staining a portion of the nitrocellulose sheet with amido black or by immunological reaction of mouse monoclonal antibody (GIN-14). For the latter, nitrocellulose sheet was blocked with BSA and reacted with GIN-14 antibody. Specific binding of monoclonal antibody was detected using peroxidase-conjugated goat anti-mouse IgG (Cappel Laboratories, Cochranville, Pa., U.S.A.) following the colour reaction with o-dianisidine and H202. Preparation ofD NA from cellular chromosome and D N A - D NA hybriclization. DNAs from cell lines were prepared as described by Sabaran et al. (1979). Cells were suspended in NTE (0. I M-NaCI, 10 mM-Tris-HC1 pH 7.4, 1 mMEDTA) containing 0.5% SDS, 500 ~tg/ml Pronase and incubated at 37 °C for 1 h (1 ml of buffer/1 x 106 cells). Cells were then extracted twice with an equal volume of NTE-saturated phenol. DNA in the aqueous phase was EtOH-precipitated and contaminating RNA was digested with RNase (100p~g/ml) and re-extracted with phenol. DNAs were digested with restriction endonuclease EcoRI (Takara Shuzo Co, Kyoto, Japan) and

A T L V - and EBV-containing B-cell lines

1783

separated by 0.8~ agarose gel electrophoresis, After gel electrophoresis the DNAs were transferred to nitrocellulose and D N A - D N A hybridization was performed. About 1 x 107 c.p.m, of nick-translated 32p-labelled probe was used for hybridization, Preparationof cellular mRNA and RNA-DNA hybridization. Cellular mRNA was prepared from ATLB 2-3, 2-21 and 2-45 using guanidine thiocyanate as described by Chirgwin et al. (1979). Cellular mRNAs were selected by oligo(dT) cellulose and 0,1 lag mRNA was separated on agarose gel electrophoresis. After electrophoresis the RNAs were transferred to nitrocellulose filters and R N A - D N A hybridization was performed as described by Thomas (1980). About 1 × 107 c.p.m. (4 × 10s &p.m./lag DNA) of nick-translated 32p-labelled probe prepared using a nick translation kit (Amersham) was used for the hybridization. DNA DNA reassociationkineticsfor detection of EB V DNA. Conditions for D N A - D N A reassociation kinetics and labelling of EBV DNA by nick translation were described previously by Hirai et aL (1981). One ng of 32p. labelled EBV (B95 8) DNA (3 × 107 c.p.m./pg) was mixed with 1 mg of the sonicated cell DNA in 0.5 ml of 0-3 MNaOH, boiled for 15 min and neutralized with HCL Then 5 M-NaC1 was added to a final concentration of 2 M in 1 ml of the mixture and samples were incubated at 67 °C. At suitable times during incubation, 0-1 ml samples were removed and stored at - 2 0 °C. Later they were assayed by single-strand-specific nuclease digestion (nuclease S1 ; Seikagaku Kogyo Co., Tokyo, Japan) to determine the amount of reassociated 32p-labelled viral DNA. The following equation was used for analysis of the data: (Co/C) l~°'ss = 1 + KCot, where C and Co are the concentrations of single-stranded 3zp-labelled viral DNA at times t and t = 0, respectively, and K is the reassociation constant (Britten & Davidson, 1976). The molecular weights of EBV DNA and cell DNA were calculated as 108 and 3.9 × 10I2, respectively. The EBV DNA used here was produced by Dr M. Nonoyama under a research project of the National Cancer Institute, NIH, Bethesda, Md., U.S.A. Blot hybridizationfor detection ofEB V DNA. Fifty lal EBV DNA or cell DNA in 0.1 M-Tris-HC1 pH 7.5, 0.007 MMgClz, 0-05 M-NaCI, 0.007 M-2-mercaptoethanol was digested with EcoRt restriction endonuclease at 37 °C for 2 h. The digested DNA was subjected to electrophoresis on a 0.5% agarose gel described previously by Hirai et al. (1981). The digested DNA fragments were immobilized on membrane filters (BA95, Schleicher & Schiill) by the transfer technique of Southern (1975). The DN A fragments immobilized on filters were incubated at 41 °C for 48 h in 50% formamide, 0.6 M-NaC1, 0.2 M-Tris--HCI pH 8.0, 0.02 M-EDTA, 0.5~ SDS, 2 × 105 to 4 × 105 c.p.m./ml of denatured 3.,P.labelled EBV DNA probe and 100 pg/ml unlabelled salmon sperm DNA in a heat-seal bag. Then filters were removed and washed three times with 2 × SSC (1 × SSC = 0-15 M-NaCL 0.015 M-sodium citrate). The filters were incubated at 41 °C overnight in 2 × SSC containing 0.5~ SDS and then washed three times with 2 × SSC, dried and exposed to Sakura X-ray film for autoradiography. RESULTS

Detection o f A TL l~:specific polypeptides p24 and p19

The experiments were performed with three clones of ATLB 2 cells, two ATLA-positive clones 2-3 and 2-21 and one negative clone 2-45, determined by indirect immunofluorescence. The results are shown in Figs. 1 and 2. When cells were metabolically labelled with [35S]methionine, an ATL-specific polypeptide p24 reacted with anti-ATLA-positive sera (Fig. 1, lanes 1, 3) but not with anti-ATLA-negative sera in clones 3 and 21 of ATLB 2 cells (Fig. 1, lanes 2, 4). Other ATLV-specific polypeptides detectable in MT-2 cell extracts (Miyoshi et al., 1981), gp68, p28, p19 and pl5 (Yamamoto et al., 1983), were not detected. By the Western blotting technique with monoclonal antibody GIN-14 an ATLA-specific polypeptide p19 was also detected in clones 3 and 21 of ATLB 2 cells (Fig. 2, lanes t, 2 respectively) (Tanaka et al., 1983). Neither p24 nor p19 were detected in the ATLA-negative clone 45 of ATLB 2 cells (data not shown). The data were in good agreement with results obtained by indirect immunofluorescence. Detection o f A T L V proviral DNA in A T L B cells T h e p r e s e n c e a n d c o n t e n t o f A T L V p r o v i r a l D N A in cellular D N A o f t h e c l o n e d A T L B 2 cells were i n v e s t i g a t e d . F o r t h i s p u r p o s e we used a 3-'P-labelled c D N A p r o b e o f A T L V w h i c h c o r r e s p o n d s to t h e U 3 R p o r t i o n o f t h e l o n g t e r m i n a l r e p e a t ( N . K o b a y a s h i et al., u n p u b l i s h e d results). W e w e r e especially i n t e r e s t e d to k n o w w h e t h e r t h e A T L A - n e g a t i v e c l o n e 2-45 was d e v o i d o f A T L V p r o v i r a l D N A . A s s h o w n in Fig. 3, all A T L B 2 c l o n e d cell~, clones 3, 21 a n d 45, contained highly conserved ATLV proviral DNA unequivocally. These ATLV provirat DNAs were d e t e c t e d in n o t only A T L A - p o s i t i v e A T L B cells b u t also in A T L A - n e g a t i v e A T L B cells.


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Fig. 1. Immunoprecipitation of ATLA-specific polypeptides from cell extracts of [3sS]methioninelabelled ATLB 2-3 (lanes 1, 2) and ATLB 2-21 (lanes 3, 4). Immunoprecipitation was carried out with anti-ATLA-positive antibody (lanes 1, 3) from an ATL patient or with anti-ATLA-negative antibody (lanes 2, 4) from a healthy adult. After adsorption to Protein A-Sepharose, precipitates were extracted and analysed by 12.5% SDS-PAGE under reducing conditions (Yamamoto et al., 1983). A calibration kit (Pharmacia) was used for molecular weight determination: phosphorylase b (94000), bovine serum albumin (67000), ovalbumin (43 000), carbonic anhydrase (30000), soybean trypsin inhibitor (20100) and e-lactalbumin (14400). Fig. 2. Western blotting analysis of proteins in cell extracts of ATLB 2-3, 2-21 and MT-2 cell lines. Proteins in extracts of I × 106 ATLB 2-3 cells (lane 1), 1 × 106 ATLB 2-21 cells (lane 2) and 2 × 104 MT-2 cells (lane 3) were separated on a 12.5°/gel blotted onto a nitrocellulose sheet and treated with G I N-14 monoclonal antibody. Peroxidase-conjugated goat anti-mouse IgG was used as an indicator of bound IgG (Towbin et al., 1979).

1785

A TL V- and EB V-containing B-cell lines 1

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Fig. 3. Detection of proviral DNA sequences of ATLV in ATLB 2-3, 2-21,2-45 and control MT-2 cells• Preparation of DNA and D N A - D N A hybridization were performed as described in Methods, Ten gg of DNA were used for each lane. Lane 1, MT-2; lane 2, ATLB 2-3; lane 3, ATLB 2-21 ; lane 4, ATLB 2-45. Dots represent the bands that hybridized the fragments corresponding to the U3R portion of ATLV long terminal repeat. HindlII-digested fragments were used as molecular size standards. Fig. 4. ATLV-specific cellular cytoplasmic mRNA from ATLB 2-3, 2-21.2-45 and control MT-2 cells. Preparation of cellular mRNA and RN A-DN A hybridization were performed as described in Methods. Lane 1, ATLB 2-3: lane 2, ATLB 2-21 ; lane 3, ATLB 2-45: lane 4, MT-2. 0,1 gg of RNA on each well was used except 0.02 gg RNA for MT-2. 28S and 18S of ribosomal RNAs were used as molecular size standards. F u r t h e r studies w i t h A T L B 5 ( A T L A - n e g a t i v e cells b y i n d i r e c t i m m u n o f l u o r e s c e n c e ) s h o w e d t h a t t h e y also p o s s e s s e d A T L V p r o v i r a l D N A ( d a t a n o t s h o w n ) .

tL~pression of ATLV-retated mRNA #z cloned cells of A T L B W e a n a l y s e d t h e e x p r e s s i o n o f A T L V - r e l a t e d p o l y ( A ) R N A in c l o n e s 3, 21 a n d 45 o f A T L B 2 cells. A s s h o w n in Fig. 4, t w o o f t h e A T L A - p o s i t i v e c l o n e 3 a n d 21 cells o f A T L B 2 c o n s e r v e d t h e

1786


ATLV-specific 35S and 26S m R N A and hybridized with a cloned D N A sequence of the ATLV genome. MT-2 cells used as control contained the ATLV-specific 35S, 32S, 26S, 24S and 20S mRNA. However, the ATLA-negative clone 45 cells did not express ATLV-specific m R N A . Thus, expression of ATLV-specific m R N A in cellular cytoplasm seems to be correlated with ATLA expression identified with GIN-14 by the indirect immunofluorescence technique or immunoprecipitation with patient sera of ATL. Analogous results were obtained from other ATLB cell lines. Detection o f E B V genomes in A TLB cells Three clones of ATLB cells were tested for their content of EBV D N A by D N A - D N A reassociation kinetics. As shown in Fig. 5, the EBNA-positive ATLB clones clearly contained EBV genomes. One of the clones (clone 3) contained about 12 copies of viral D N A per cell. However, the EBV-negative MT-1 cells (Miyoshi et al., 1980) used as a negative control contained no measurable amount of EBV DNA. The reassociation of 32p-labelled EBV D N A and ATLB cell D N A proceeded linearly without a break (Fig. 5), suggesting that most of the viral DNA sequences are present in ATLB cells. Restriction endonuclease cleavage patterns o f EB V D N A in A TLB cells

To analyse the EBV genome structure in ATLB cells, the sizes of EcoRI cleavage fragments of EBV D N A in ATLB clone 3 cells were compared with those of B95-8 (EBV) D N A by their electrophoretic mobilities in agarose gels. The viral D N A fragments identified by hybridization to 32p-labelled B95-8 virion D N A were reported in some detail by Given & Kieff (1978). Therefore, their nomenclature for the EcoRI fragments of B95-8 D N A was adopted in this report. The EcoRI fragments that co-migrate with B95-8 were assumed to be identical and occupy the same position of the EBV physical map. Fig. 6 shows that most of the EcoRI fragments of EBV D N A in ATLB clone 3 cells were similar in size to fragments of B95-8 virion DNA. DISCUSSION The presence of ATLA and EBNA concomitantly in the series of ATLB cell lines established from ATL patients was first shown by the use of indirect immunofluorescence. In this study, we have further analysed these cell lines and their subclones. The data show that ATLB cells possess (i) ATLA-specific polypeptides in ATLA-positive clones, (ii) ATLV proviral D N A and EBV DNA, and (iii) ATLV-specific m R N A in ATLA-positive clones. Thus, ATLB cell lines are dually infected with ATLV and EBV. However, this feature of dual infection is not unique to ATLB lines since similar cell lines have been generated experimentally, for example by co-cultivation of the B-cell-rich fraction from PBL of healthy adults with X-irradiated MT-2 cells (Okada et aI., 1984). Analogous cell lines possessing both ATLV- and EBV-like agents have been established also from African green monkeys and Japanese monkeys by co-cultivation techniques (N. Yamamoto et al., unpublished results), The data presented here show that Blymphocytes are susceptible to ATLV infection. These results differ from the studies with HTLV showing the development of an EBV-transformed line from an HTLV-infected patient, which is HTLV-negative (Gallo et al., 1982). Further studies are required to clarify this discrepancy. ATLV-specific polypeptides p24 and p19 were recognized from ATLB 2-3 and 2-21 cell extracts. The p24 and p19 are considered as core polypeptides of ATLV (Kalyanaraman et al., 1981; Yamamoto & Hinuma, 1982; Yamamoto et al., 1983), whereas other ATLV-specific polypeptides detectable in MT-2 cell extracts such as gp68, p28 and p15 were not detectable in the ATLB cells. The Western blotting technique revealed that ATLB 2-3 and 2-21 were only positive for p19 but not for p28 both of which were detectable simultaneously in MT-2 cell extracts with a mouse monoclonal antibody (GIN-14). It is not yet clear whether this shows the lack of gp68, p28 and pl 5 in ATLB lines or simply reflects the quantitative difference in the amount of such polypeptides between MT-2 and ATLB cell lines. Analysis of the expression of ATLV-related poly(A) RNA in cloned cells of ATLB 2 provided

1787

A T L V- and E B V-containing B-cell lines I

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Fig. 5. D N A D N A reassociation kinetics of 32p-labelled EBV D N A with D N A of ATLB cells. D N A - D N A reassociation kinetics were examined as described in Methods. One ng of 32p-labelled EBV (B95-8) D N A prepared by nick translation was mixed with the following D N A s in 1 ml: 1 mg Molt cell D N A (Srivastava & Minowada, 1973) (C)); 0.026 ~tg cold EBV D N A (one genome/cell) and 1 mg Molt cell D N A (O); 0-26 ~tg cold EBV D N A (ten genomes/cell) and 1 mg Molt cell D N A ( A); 0.5 mg ATLB 2-21 cell D N A and 0.5 mg Molt cell D N A (A); 0-5 mg ATLB 2-3 cell D N A and 0-5 mg Molt cell D N A ( I ) ; 0.5 mg ATLB 2-23 cell DNA and 0.5 mg Molt cell D N A (D); 1 mg MT-I cell D N A (V). Fig. 6. Blot hybridization of 32p-labelled EBV D N A to blots of EcoRI D N A fragments of the B95-8 virion and ATLB 2-3 cell DNAs. EBV (B95-8) D N A (0-1 ~tg) (lane 1) or D N A (20 ~tg) extracted from ATLB 2-3 cells (lane 2) was digested with EcoRI. The products were separated by electrophoresis in agarose gels and transferred to membrane filters. 32p-labelled EBV (B95-8) D N A was hybridized to the filter strips. The filters were washed and autoradiographed. Designations of EcoRl fragments of EBV (B95-8) D N A are taken from Heller et aL (1981) and indicated next to the corresponding restriction fragment band.

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Y. K O Y A N A G I A N D O T H E R S

good correlation with ATLA expression. We identified the ATLV-specific 35S, 30S and 26S m R N A in ATLA-positive B-cells, ATLB 2-3 and 2-21, but not in the ATLA-negative B-cells, ATLB 2-45. Preliminary studies showed that in other ATLA-positive cell lines, the ATLVspecific 35S and 26S m R N A s were commonly detected. In addition to 35S and 26S m R N A smaller sized m R N A s were detected in respective cells. For example, in MT-2 cells mainly 24S and a small amount of 32S m R N A and in H A M A cells, a T-cell line established from an ATL patient, mainly 25S mRNA. The 35S RNA may be considered as a helper type R N A genome from its size (Yoshida et at., 1982; N. Kobayashi et al., unpublished results). Because of the failure to detect 26S RNA from MT-2 virion RNA, we can only postulate that the detectable 26S RNA was processed from 35S R N A (N. Kobayashi et al., unpublished results). Further studies are required to clarify these possibilities. In the Southern blotting studies, we detected the ATLV proviral D N A in not only ATLApositive ATLB cells but also in ATLA-negative cells. All ATLB cell lines examined to date and OKA(B) cell lines established by co-cultivation techniques possessed ATLV proviral D N A whether or not they expressed ATLA. Whether the proviruses are complete or defective remains to be determined. In various ATLB cell lines, EBV genomes were really identified and most viral D N A sequences were detected. The E c o R I cleavage maps showed that most of the E c o R I fragments in ATLB 2-3 were similar in size to those of B95-8 virion DNA. Previously, it was reported that the EcoRI cleavage patterns obtained from the different EBV strains were very similar with some variabilities (Bornkamm et al., 1980; Heller et al., 1981). The B95-8 E c o R I - C fragment is smaller than that of the EBV DNA from ATLB 2-3 cells. This could be due to the fact that B95-8 viral DNA has a large deletion at the location of EcoRI-C fragment on the EBV genome map (Given & Kieff, 1978: Raab-Traub et al., 1978). The results suggest that the EBV D N A s of B%5-8 and ATLB cells are largely co-linear in organization. However, it is not known whether the intracellular EBV D N A in ATLB 2-3 cells exists as a circular episome or in an integrated state. It is known that EBV(+) B-cell lines are spontaneously established from the patients with infectious mononucleosis and healthy seropositives. This suggests that E B V ( + ) B-lymphocytes per se have the capacity to grow permanently when transferred in vitro. Therefore, it is very likely that A T L V ( + ) B-cells have the same capability. Data presented in this study, however, clearly demonstrated that ATLV formerly identified only in T-cells could also infect B-cells. We thank Dr James R. Blakeslee, Jr., The Ohio State University, for revision of this manuscript. This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan. REFERENCES BONNER, W. M. & LASKEY, R, A. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. European Journal of Biochemistry 46, 83 88. BORNKAMM,G. W~, DEL1US,n., ZIMBER, U., HUDEWENTZ, J. & EPSTEIN, M. A. (1980). Comparison of Epstein Barr virus strains of different origin by analysis of the viral D N A s . Journal of Virology 35, 603-618. BRITTEN, R. J. & DAVIDSON, E. H. (t976). Studies on nucleic acid reassociation kinetics: empirical equations describing D N A reassociation. Proceedings of the National Academy of Sciences, U.S.A. 73, 415-419. CHIRGWIN, J. M.. PRZYBYLA, A. E., MACDONALD, R. L & RUTLER, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. DEINHARDT, F. & DEINHARDT,J. ( 1979). 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(Received 23 February t984)