Proc. Natl. Acad. Sci. USAVol. 81, pp. 3526-3528, June 1984Medical Sciences

Evidence of extrachromosomal forms of hepatitis B viral DNA in abone marrow culture obtained from a patient recently infectedwith 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 Schoolof Public Health, Boston, MA 02115

Communicated by Hans Popper, February 7, 1984

ABSTRACT A cell culture that produces Dane-like parti-cles was initiated from a bone marrow aspirate of an acutehepatitis B patient. By using Southern blot analysis and arecombinant hepatitis B virus (HBV) DNA plasmid probe, ex-trachromosomal forms of HBV DNA were detected. The twoforms of HBV DNA migrate as a closed circular 2.2-kb formand an open circular 3.9-kb form. There was no evidence ofHBV DNA integration into the host genome.

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Research on the biology and replication of human hepatitis Bvirus (HBV) has been hampered by a lack of cell culture sys-tems for this virus (1-2). Although cell lines exist that con-tain integrated copies of HBV DNA, these cell lines do notproduce complete virus (3-9). Recently, a cell culture wasestablished from a bone marrow aspirate of a patient with anacute HBV infection, who was seriologically positive for thehepatitis B surface antigen (HBsAg) and HBVe antigen andhad antibodies to hepatitis virion core proteins (HBcAg).The cell culture, designated RAC/BM, contains a fraction ofcells that produces virion-like Dane particles and is positiveboth for the HbsAg and the HbcAg (10). The continuedexpression of both antigens suggests that this culture may bepersistently infected with HBV. If this is the case, then theRAC/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 noevidence of integration of HBV DNA into the host genomeafter 1 year in culture.

MATERIALS AND METHODSDNA 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 obtainedfrom Dr. Jack Little. DNA was extracted in lysis buffer (10mM Tris HCl, pH 7.4/10mM NaCl/2 mM Na2EDTA) andincubated at 37°C with proteinase K (500 ,g ml-) and 0.5%NaDodSO4 for 2 hr. After proteinase K treatment, Na-DodSO4 was added to a final concentration of 1%, and thecellular DNA was extracted twice with phenol saturatedwith 20 mM Tris HCI (pH 7.4) and once with chloroform/isoamyl alcohol, 24:1 (vol/vol). The DNA was precipitatedwith 2 vol of ethanol overnight at -20°C. The precipitate wasresuspended in the lysis buffer and treated with RNase A at100 ,g-ml-1 for 1 hr at 37°C. The phenol and chloroform/isoamyl alcohol extractions were repeated, and the DNAwas precipitated with ethanol and resuspended in 10 mMTris HCI, pH 7.4/1 mM EDTA.

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FIG. 1. Southern blot analysis of RAC/BM culture. CellularDNA (10 ,ug) was digested with HindIll or EcoRI. DNA sampleswere fractionated in 0.8% agarose gel, transferred to nitrocellulosemembrane by the method of Southern (12-13), and hybridizedagainst the 32P-labeled probe of cloned pAOl HBV DN (11). Lanes:1 and 2, cellular DNA PLC/PRF/5 digested with HindIII andEcoRI, respectively; 3, normal human fibroblast DNA digested withEcoRI; 4, RAC/BM DNA digested with EcoRI; 5, undigestedRAC/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. Elec-trophoresis was performed in 0.8% agarose gels at 30 V for18 hr. DNA was transferred to nitrocellulose filter by theprocedure of Southern (11) as modified by Whal (12). Theplasmid pAOl-HBV used as a probe was labeled with the

Abbreviations: HBV: hepatitis B virus, HBsAg: hepatitis B surfaceantigen, HBcAg: hepatitis B core antigen.

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Proc. NatL. Acad. Sci. USA 81 (1984) 3527

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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 anEcoRI 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 humanfibroblast 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 pro-cedure (13). The specific activity of the probe was 5-6 x 108cpm/pug. The filters were prehybridized at 680C for 4 hr in5 x SCC NaCl/Cit (1 x NaCl/Cit = 0.15 M NaCl/0.015 M Nacitrate, pH 7) containing 0.5% NaDodSO4, 5x Denhardt'ssolution (0.02% polyvinylpyrrolidone/0.02% Ficoll/0.2%bovine serum albumin), and denatured salmon sperm DNAat 100 tkg ml-l. The hybridization was camed out at 680C for24-36 hr in Sx NaCl/Cit containing 0.5% NaDodSO4, SxDenhardt's solution, 100 pg/ml of denatured salmon spermDNA and denatured probe (1.107 cpm). After hybridizationthe filters were washed at room temperature in 2 x NaCl/Citcontaining 0.5% NaDodSO4 and rinsed in 0.2x NaCl/Citcontaining 0.5% NaDodSO4 for 2 hr at 680C. Filters weredried and exposed at -80'C.

RESULTSFor these experiments, total DNA was extracted from thecells. The molecular forms of DNA homologous to HBV inRAC/BM cells was investigated. The DNA was purified andanalyzed for the presence of HBV sequences by Southernblot analysis. A high-specific-activity 32P-labeled DNAprobe (-5 x 108 cpm/jxg) was synthesized by nick-transla-tion from a plasmid that contained a complete integrated

copy of the HBV genome (14). The number of cells that wereHBsAg-positive in immunofluorescence assays varied from15% to <5% of the total cell population.An autoradiogram of total, uncleaved DNA extracted

from the culture showed that two molecular-size classes ofDNA homologous to HBV DNA were present in the RAC/BM culture (Fig. 1, lane 5). These species migrated with theapparent length of 3.9 and 2.2 kilobases (kb), as determinedby comparison with DNA fragments of known length includ-ed on the same gel. No such species were detected in DNAextracted from normal human fibroblasts (Fig. 1, lane 3). Nohybridization with pBR322 DNA probe was detected in anyof these samples. The presence of discrete-size, low molecu-lar weight species in undigested DNA ofRAC/BM cells indi-cates an extrachromosomal origin of the HBV-related se-quences. No such species were identified in the undigestedDNA extracted from the human hepatoma cell linesPLC/PRF/5 and Hep3B (not shown) that contained integrat-ed copies of HBV DNA (15-17). In the case of thePLC/PRF/5 line, all sequences homologous to HBV migrat-ed at the top of the gel with the high molecular weight DNA,although there is a single report of unintegrated DNA in thiscell line (18).To determine if the RAC/BM culture contained integrated

HBV DNA molecules, the DNA was digested with the re-

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Medical Sciences: Elfassi et aL

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3528 Medical Sciences: Elfassi etaLP

striction enzymes HindIII and EcoRI. These enzymes were

chosen because they permit discrimination between integrat-ed and nonintegrated HBV DNA. The enzyme HindIII doesnot cleave the HBV genome, and EcoRI is reported to cutmost strains of HBV DNA at a single site (19, 20). WhenDNA extracted from the RAC/BM culture was digested withHindIII, the same species as the undigested DNA 3.9- and2.2-kb molecules were observed (Fig. 2, lane 9; Fig. 1, lane5). In contrast, HindIII digestion of the hepatoma cell linePLC/PRF/5 yielded at least six different bands ranging insize from 4.2 to >23 kb (Fig. 1, lane 1) that were homologousto HBV DNA. This is consistent with the earlier reports ofmultiple integration sites of HBV DNA in this cell line (15-17).

Digestion of the DNA extracted from RAC/BM culturewith the restriction enzyme EcoRI yielded a single DNAfragment 3.2 kb long (Fig. 1, lane 4; Fig. 2, lane 8), the lengthof the HBV genome itself. The observation that the twoHBV molecules observed in the undigested DNA sample(Fig. 1, lane 5) were converted to a single species that mi-grated with an apparent length of 3.2 kb upon digestion withEcoRI suggests that both the 3.9- and 2.2-kb molecules arephysical isomers of the HBV genome. These two speciespresumably migrate as the closed circular (2.2 kb) and opencircular (3.9 kb) forms of HBV DNA. No such species wereobserved upon digestion of total human DNA with EcoRI(Fig. 1, lane 3). From these experiments we conclude thatsequences homologous to HBV DNA that are present in theRAC/BM culture are present predominantly in extrachro-mosomal forms.The amount of DNA homologous to HBV DNA in the

RAC/BM culture was estimated by comparison of the inten-sity of the 4.3-kb band that corresponds to one of the inte-grated copies present in the PLC/PRF/5 genome to the in-tensity of the bands in the undigested sample of RAC/BMDNA. This comparison assumes that the band in thePLC/PRF/5 DNA represents a unique copy and that the ex-tent of hybridization of the probe to each species is compara-ble. With these assumptions, we estimate that in the experi-ment shown in Fig. 2, there was approximately one genomeequivalent to HBV DNA per cell in the RAC/BM culture.We note that there was a variation in the amount of thesespecies detected from different DNA extractions. This isprobably related to a fluctuation observed in the fraction ofcells that expressed the HBV antigens in this culture.

DISCUSSION

The observation that the RAC/BM culture contains primari-ly, and perhaps exclusively, unintegrated DNA differs fromwhat has been found previously for established human hepa-toma cell lines. In such cases, integrated forms of HBVDNA were found (3-8). The culture also differs in that bothHBcAg and HBsAg are expressed, whereas only fIBsAg isexpressed in the hepatoma cell lines. Extrachromosomal cir-cular DNA forms of human HBV have been reported previ-ously in the livers of chimpanzees that are chronic HBV car-

riers (21). As in the case of the bone marrow cell culturereported here, there was no evidence of integration of HBVDNA in the chimpanzee liver cells. More recently, other lab-oratories have observed circular extrachromosomal forms ofHBV DNA in the livers of Peking ducks (22, 23) and groundsquirrels (24) infected with the corresponding hepadna virus-es. In these cases, the circular forms ofDNA are associatedwith active replication of the HBV. The observation that the

RAC/BM culture produces virion-like Dane particles, ex-presses both HBsAg and HBcAg, and contains extrachro-mosomal forms ofDNA suggests that active replication maybe in progress in this culture. However, we cannot rule outthe possibility that HBV DNA replicates in an extrachromo-somal 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 con-tains extrachromosomal forms of HBV genome raises thepossibility that the bone marrow, and perhaps more particu-larly lymphoid or myeloid blast cells, may be a natural targetfor HBV infection. Infection of bone marrow cells during thenatural course of HBV infection may have direct implica-tions for the pathogenesis of HBV in man.

The authors are grateful to Dr. J. Summers for providing the recom-binant plasmid pAOl-HBV and for helpful discussions. The authorsthank Dr. J. Sodroski, Dr. M. Trus, and Dr. J. Mullins for theirhelpful discussion. This work was supported by CA29294 to W.H.E.E. is the recipient of a Leukemia Society of America Fellowshipaward.

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