cells that produces virion-like Dane particles and is positive both for the HbsAg ..... Twist, E. M., Clark, H. F., Aden, D. P., Knowles, B. B. &. Plotkin, S.A. (1981) J.
Proc. Natl. Acad. Sci. USA Vol. 81, pp. 3526-3528, June 1984 Medical Sciences
Evidence of extrachromosomal forms of hepatitis B viral DNA in a bone marrow culture obtained from a patient recently infected with hepatitis B virus (human bone marrow/Southern blot analysis)
EMILE ELFASSI*, JEAN-LOUP ROMET-LEMONNEt, MYRON ESSEXt, MARY FRANCES-MCLANEt, AND WILLIAM A. HASELTINE*t *Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School, Boston, MA 02115; and tDepartment of Cancer Biology, Harvard School of Public Health, Boston, MA 02115
Communicated by Hans Popper, February 7, 1984
A cell culture that produces Dane-like partiABSTRACT cles was initiated from a bone marrow aspirate of an acute hepatitis B patient. By using Southern blot analysis and a recombinant hepatitis B virus (HBV) DNA plasmid probe, extrachromosomal forms of HBV DNA were detected. The two forms of HBV DNA migrate as a closed circular 2.2-kb form and an open circular 3.9-kb form. There was no evidence of HBV DNA integration into the host genome.
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Research on the biology and replication of human hepatitis B virus (HBV) has been hampered by a lack of cell culture systems for this virus (1-2). Although cell lines exist that contain integrated copies of HBV DNA, these cell lines do not produce complete virus (3-9). Recently, a cell culture was established from a bone marrow aspirate of a patient with an acute HBV infection, who was seriologically positive for the hepatitis B surface antigen (HBsAg) and HBVe antigen and had antibodies to hepatitis virion core proteins (HBcAg). The cell culture, designated RAC/BM, contains a fraction of cells that produces virion-like Dane particles and is positive both for the HbsAg and the HbcAg (10). The continued expression of both antigens suggests that this culture may be persistently infected with HBV. If this is the case, then the RAC/BM culture should contain HBV DNA. Here we report that the RAC/BM culture contains a low level of extrachromosomal HBV DNA and that there is no evidence of integration of HBV DNA into the host genome after 1 year in culture.
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MATERIALS AND METHODS DNA Extraction. DNA was prepared from 108 cells. Cells used included a human hepatoma cell line, PLC/PRF/5 (4); a human lymphoblastoid cell culture, RAC/BM culture (10); and a normal human fibroblast designated 1522 and obtained from Dr. Jack Little. DNA was extracted in lysis buffer (10 mM Tris HCl, pH 7.4/10mM NaCl/2 mM Na2EDTA) and incubated at 37°C with proteinase K (500 ,g ml-) and 0.5% NaDodSO4 for 2 hr. After proteinase K treatment, NaDodSO4 was added to a final concentration of 1%, and the cellular DNA was extracted twice with phenol saturated with 20 mM Tris HCI (pH 7.4) and once with chloroform/ isoamyl alcohol, 24:1 (vol/vol). The DNA was precipitated with 2 vol of ethanol overnight at -20°C. The precipitate was resuspended in the lysis buffer and treated with RNase A at 100 ,g-ml-1 for 1 hr at 37°C. The phenol and chloroform/ isoamyl alcohol extractions were repeated, and the DNA was precipitated with ethanol and resuspended in 10 mM Tris HCI, pH 7.4/1 mM EDTA.
FIG. 1. Southern blot analysis of RAC/BM culture. Cellular DNA (10 ,ug) was digested with HindIll or EcoRI. DNA samples were fractionated in 0.8% agarose gel, transferred to nitrocellulose membrane by the method of Southern (12-13), and hybridized against the 32P-labeled probe of cloned pAOl HBV DN (11). Lanes: 1 and 2, cellular DNA PLC/PRF/5 digested with HindIII and EcoRI, respectively; 3, normal human fibroblast DNA digested with EcoRI; 4, RAC/BM DNA digested with EcoRI; 5, undigested RAC/BM DNA. Sizes are shown in kb.
Enzyme Digestion. Digestion with restriction endonucle-
ases EcoRI, HindIII, and BamHI (New England Biolabs) was carried out according to the manufacturer's specifica-
tions. Radiolabeling of Probe and Southern Blot Analysis. Electrophoresis was performed in 0.8% agarose gels at 30 V for 18 hr. DNA was transferred to nitrocellulose filter by the procedure of Southern (11) as modified by Whal (12). The plasmid pAOl-HBV used as a probe was labeled with the
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Abbreviations: HBV: hepatitis B virus, HBsAg: hepatitis B surface antigen, HBcAg: hepatitis B core antigen. 3526
Medical Sciences: Elfassi et aL
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Proc. NatL. Acad. Sci. USA 81 (1984)
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FIG. 2. Hybridization of 32P-labeled HBV DNA probe to a BamHI digest of PLC/PRF/5 DNA (10 yg to 156 ng) (lanes 1-7); 10 lig of an EcoRI digest of RAC/BM DNA (lane 8), 10 tig of HindIII-digested RAC/BM DNA (lane 9); and 10 Mg of a BamHI digest of normal human fibroblast DNA (lane 10). Sizes are shown in kb.
four [32p]-dNTPs (200 pmol of each (with specific activity, 3200 Ci mmol-F; 1 Ci = 37 GBq) by the nick-translation procedure (13). The specific activity of the probe was 5-6 x 108 cpm/pug. The filters were prehybridized at 680C for 4 hr in 5 x SCC NaCl/Cit (1 x NaCl/Cit = 0.15 M NaCl/0.015 M Na citrate, pH 7) containing 0.5% NaDodSO4, 5x Denhardt's solution (0.02% polyvinylpyrrolidone/0.02% Ficoll/0.2% bovine serum albumin), and denatured salmon sperm DNA at 100 tkg ml-l. The hybridization was camed out at 680C for 24-36 hr in Sx NaCl/Cit containing 0.5% NaDodSO4, Sx Denhardt's solution, 100 pg/ml of denatured salmon sperm DNA and denatured probe (1.107 cpm). After hybridization the filters were washed at room temperature in 2 x NaCl/Cit containing 0.5% NaDodSO4 and rinsed in 0.2x NaCl/Cit containing 0.5% NaDodSO4 for 2 hr at 680C. Filters were dried and exposed at -80'C.
RESULTS For these experiments, total DNA was extracted from the cells. The molecular forms of DNA homologous to HBV in RAC/BM cells was investigated. The DNA was purified and analyzed for the presence of HBV sequences by Southern blot analysis. A high-specific-activity 32P-labeled DNA probe (-5 x 108 cpm/jxg) was synthesized by nick-translation from a plasmid that contained a complete integrated
of the HBV genome (14). The number of cells that were HBsAg-positive in immunofluorescence assays varied from 15% to 23 kb (Fig. 1, lane 1) that were homologous to HBV DNA. This is consistent with the earlier reports of multiple integration sites of HBV DNA in this cell line (1517). Digestion of the DNA extracted from RAC/BM culture with the restriction enzyme EcoRI yielded a single DNA fragment 3.2 kb long (Fig. 1, lane 4; Fig. 2, lane 8), the length of the HBV genome itself. The observation that the two HBV molecules observed in the undigested DNA sample (Fig. 1, lane 5) were converted to a single species that migrated with an apparent length of 3.2 kb upon digestion with EcoRI suggests that both the 3.9- and 2.2-kb molecules are physical isomers of the HBV genome. These two species presumably migrate as the closed circular (2.2 kb) and open circular (3.9 kb) forms of HBV DNA. No such species were observed upon digestion of total human DNA with EcoRI (Fig. 1, lane 3). From these experiments we conclude that sequences homologous to HBV DNA that are present in the RAC/BM culture are present predominantly in extrachromosomal forms. The amount of DNA homologous to HBV DNA in the RAC/BM culture was estimated by comparison of the intensity of the 4.3-kb band that corresponds to one of the integrated copies present in the PLC/PRF/5 genome to the intensity of the bands in the undigested sample of RAC/BM DNA. This comparison assumes that the band in the PLC/PRF/5 DNA represents a unique copy and that the extent of hybridization of the probe to each species is comparable. With these assumptions, we estimate that in the experiment shown in Fig. 2, there was approximately one genome equivalent to HBV DNA per cell in the RAC/BM culture. We note that there was a variation in the amount of these species detected from different DNA extractions. This is probably related to a fluctuation observed in the fraction of cells that expressed the HBV antigens in this culture.
DISCUSSION The observation that the RAC/BM culture contains primarily, and perhaps exclusively, unintegrated DNA differs from what has been found previously for established human hepatoma cell lines. In such cases, integrated forms of HBV DNA were found (3-8). The culture also differs in that both HBcAg and HBsAg are expressed, whereas only fIBsAg is expressed in the hepatoma cell lines. Extrachromosomal circular DNA forms of human HBV have been reported previously in the livers of chimpanzees that are chronic HBV carriers (21). As in the case of the bone marrow cell culture reported here, there was no evidence of integration of HBV DNA in the chimpanzee liver cells. More recently, other laboratories have observed circular extrachromosomal forms of HBV DNA in the livers of Peking ducks (22, 23) and ground squirrels (24) infected with the corresponding hepadna viruses. In these cases, the circular forms of DNA are associated with active replication of the HBV. The observation that the
(1984)
RAC/BM culture produces virion-like Dane particles, expresses both HBsAg and HBcAg, and contains extrachromosomal forms of DNA suggests that active replication may be in progress in this culture. However, we cannot rule out the possibility that HBV DNA replicates in an extrachromosomal form rather than spreading through infection. This report of a human culture that expresses both HBsAg and HBcAg, produces a virion-like Dane particle, and contains extrachromosomal forms of HBV genome raises the possibility that the bone marrow, and perhaps more particularly lymphoid or myeloid blast cells, may be a natural target for HBV infection. Infection of bone marrow cells during the natural course of HBV infection may have direct implications for the pathogenesis of HBV in man. The authors are grateful to Dr. J. Summers for providing the recombinant plasmid pAOl-HBV and for helpful discussions. The authors thank Dr. J. Sodroski, Dr. M. Trus, and Dr. J. Mullins for their helpful discussion. This work was supported by CA29294 to W.H. E.E. is the recipient of a Leukemia Society of America Fellowship award. 1. Zuckerman, A. J. & Howard, C. R. (1979) Hepatitis Viruses of Man (Academic, London). 2. Tiollais, P., Charnay, P. & Vyas, G. N. (1981) Science 213, 406-411. 3. Alexander, J. J., Bey, E. M., Geddes, E. W. & Lecatsas, G. S. Afr. Med. J. 50, 2124-2128. 4. Macnab, G. M., Alexander, J. J., Lecatsas, G., Bey, E. M. & Urbanowicz, J. M. (1976) Br. J. Cancer 34, 509-515. 5. Skelly, S., Copeland, J. A., Howard, C. R. & Zuckerman, A. J. (1976) Nature (London) 282, 617-618. 6. Knowles, B. B., Howe, C. C. & Aden, D. P. (1980) Science 209, 497-499. 7. Aden, D. P., Fogel, A., Plotkin, S., Damjonov, I. & Knowles, B. B. (1979) Nature (London) 282, 615-617. 8. Koike, K., Kobayashi, M., Mizusawa, H., Yoshida, E., Yaginuma, K. & Taira, M. (1983) Nucleic Acids Res. 11, 53915402. 9. Gough, N. M. & Murray, K. (1982) J. Mol. Biol. 162, 43-67. 10. Romet-Lemonne, J. L., McLane, M. F., Elfassi, E., Haseltine, W. A., Azocar, J. & Essex, M. (1983) Science 221, 667669. 11. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. 12. Whal, G. M., Stern, M. & Stark, G. R. (1979) Proc. Natl. Acad. Sci. USA 76, 3683-3687. 13. Rigby, P. W., Dieckman, J. M., Rhodes, C. & Berg, P. (1977) J. Mol. Biol. 113, 237-251. 14. Cummings, I. W., Browne, J. K., Salser, W. A., Tyler, G. V., Snyder, R. L., Smolec, J. M. & Summers, J. (1983) Proc. Natl. Acad. Sci. USA 77, 1842-1846. 15. Edman, J. C., Gray, P., Valenzuela, P., Rall, L. B. & Rutter, W. J. (1980) Nature (London) 286, 535-537. 16. Twist, E. M., Clark, H. F., Aden, D. P., Knowles, B. B. & Plotkin, S. A. (1981) J. Virol. 37, 239-243. 17. Brechot, C., Purcell, C., Louise, A., Rain, B. & Tiollais, P. (1977) Nature (London) 236, 533-535. 18. Zaslavsky, V., Marquardt, O., Wong, T. K. & Hofschneider, P. H. (1980) J. Gen. Virol. 51, 341-349. 19. Siddiqui, A., Sattler, F. & Robinson, W. (1979) Proc. Natl. Acad. Sci. USA 76, 4664-4668. 20. Charnay, P., Purcell, C., Fritzch, A., Louise, A. & Tiollais, P. (1979) Proc. Natl. Acad. Sci. USA 76, 2222-2226. 21. Ruiz-Opadzo, N., Chakroborty, P. R. & Shafritz, D. A. (1983) Cell 29, 129-138. 22. Summers, J. & Mason, W. S. (1982) Cell 29, 403-415. 23. O'Connel, A. P., Urban, M. K. & London, W. T. (1983) Proc. Natl. Acad. Sci. USA 80, 1703-1706. 24. Weiser, B., Ganem, D., Seeger, C. & Varmus, H. E. (1983) J. Virol. 48, 1-9.
