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Hepatitis B virus replication and viral antigen synthesis in hepatocyte lines derived from normal human liver1
Virus Research 52 (1997) 177 – 182
Hepatitis B virus replication and viral antigen synthesis in hepatocyte lines derived from normal human liver1 Jasper zu Putlitz 2,a, Eve A. Roberts b, Stefan Wieland a, Yumi Kono 3,b, Hubert E. Blum a,* a
Department of Internal Medicine II, Uni6ersity Hospital Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany b The Hospital for Sick Children Research Institute, Uni6ersity of Toronto, Toronto, Ont., Canada Received 9 May 1997; received in revised form 6 September 1997; accepted 15 September 1997
Abstract Transient transfection and in vitro infection experiments were performed to characterize replication and antigen synthesis of the hepatitis B virus (HBV) in human hepatocyte lines HH29 and HHY41, derived from normal liver tissue. These liver cell lines are capable of supporting HBV replication and gene expression at levels similar to the human hepatoma cell line HuH-7. Strikingly, a very tight adhesion of HBV to the outer cell membrane of HH29 and HHY41 was observed under conditions that removed HBV to undetectable levels from HuH-7 hepatoma cells. However, no productive HBV infection could be established in these cells as determined by the absence of viral transcripts and de novo antigen synthesis. In conclusion, the human hepatocyte cell lines HH29 and HHY41 may be useful to study important aspects of late steps in the replication of HBV, but appear to lack certain cellular components that play a pivotal role during early steps of the viral life cycle. © 1997 Elsevier Science B.V. Keywords: Hepatitis B virus; Hepatocytes; Transfection; Receptor
* Corresponding author. Tel.: +49 761 2703403; fax: + 49 761 2703610. 1 Presented in part at the 1996 Annual Meeting of the American Association for the Study of Liver Diseases and published in abstract form (Hepatology 1996;24(4):224A). 2 Present address: Molecular Hepatology Laboratory, MGH Cancer Center, Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA. 3 Present address: Department of Pediatrics, Faculty of Medicine, Tottori University, Yonago, Japan.
Chronic hepatitis B virus (HBV) infection is a major public health problem. Although the replication strategy of HBV is fairly well understood (Ganem and Varmus, 1987), little is known about the cellular receptor for HBV and early events in the viral life cycle, due to the lack of an efficient in vitro infection system. Previously established hepatoma cell lines, such as HuH-7 (Nakabayashi et al., 1982) and HepG2 (Sells et al., 1987), can be
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infected in vitro only at very low efficiency (Mabit et al., 1994), but support the replication of HBV when plasmid-encoded, replication-competent overlength constructs of HBV DNA are transfected into these cells. We investigated whether the human hepatocyte lines HH29 and HHY41 support the replication of HBV. Because these cells were derived from normal liver tissue rather than from a hepatocellular carcinoma, we reasoned that they might have retained cellular components that are essential during viral entry and/or early phases of the viral life cycle in the host cell. We therefore attempted to infect these cells in vitro with HBV. The human hepatocyte cell lines HH29 and HHY41 were previously established from surgically resected, normal liver tissue at The Hospital for Sick Children Research Institute, Toronto, Canada. HH29 was established by co-culture with rat liver epithelial cells (RLEC) as described (Roberts et al., 1994). HHY41 was established by prolonged culture between two layers of hydrated collagen (collagen gel sandwich) in a medium enriched with growth factors (Kono et al., 1995). These cell lines exhibit morphological and functional characteristics of differentiated hepatocytes. They retain glucose-6-phosphatase activity, produce albumin and other proteins typical of hepatocytes, and maintain various active cytochromes P-450. HH29 and HHY41 were grown in Dulbecco’s modification of Eagle’s medium (DMEM)/F12 (Life Technologies, Gaithersburg, MD) supplemented with 8% FCS, insulin 10 mg/ml, hydrocortisone 10 − 6 M, linoleic acid/bovine serum albumin 0.5 mg/ml/0.05 mg/ml, thyrotropin-releasing hormone 10 − 6 M, selenous acid 10 − 7 M (all supplements from Sigma, St. Louis, MO) and 1% penicillin/streptomycin stock solution (5000 U/ml penicillin G sodium/5000 mg/ml streptomycin). The cell line HuH-7 (Nakabayashi et al., 1982), derived from a human hepatocellular carcinoma, was grown in Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies), supplemented with 8% fetal calf serum (FCS), 1% non-essential amino acid solution (Life Technologies) and 1% penicillin/streptomycin stock solution (5000 U/ml penicillin G sodium/5000 mg/ml streptomycin; Life Technologies).
The human hepatocyte lines HH29 and HHY41 were transiently transfected using a modified calcium phosphate precipitation protocol (Chen and Okayama, 1987), routinely using 10 mg of a headto-tail dimer of wild-type HBV, subtype adw (ADW-HTD; GenBank™ accession number X02763; Blum et al., 1991) plus 1 mg of reporter plasmid (pCAT-Control; Promega, Madison, WI) per 100-mm plate. The variation in transfection efficiencies was assessed by CAT assay as described (Sleigh, 1986). The construct ADW-HTD allows for the replication of HBV. As a consequence of the synthesis of viral DNA and proteins, viral antigens are released into the cell culture supernatant, and nucleocapsid-associated replication products can be detected in cellular lysates. As positive control for virus replication, transfections were performed in parallel with the human hepatoma cell line HuH-7 which is known to support the replication of HBV. After incubation for 5 days, cellular lysates were analyzed for the presence of HBV nucleocapsid-associated nucleic acid. For the preparation of nucleocapsid-associated HBV DNA, transfected cells were lysed in 50 mM Tris (pH 8.0)/1 mM EDTA/1% Nonidet P-40. The lysate was centrifuged at 10 000× g for 5 min at room temperature. After the addition of CaCl2 and MgCl2 to a final concentration of 10 mM each, the supernatant was incubated with 20 U/ml of DNase I (Boehringer Mannheim) and micrococcal nuclease (final concentration 150 U/ ml; Pharmacia Biotech) for 2 h at 37°C. Next, EDTA (final concentration, 20 mmol/l), 10% sodium dodecyl sulfate (final concentration, 1%) and proteinase K (final concentration, 1 mg/ml; Promega) was added, followed by incubation for 12–16 h at 37°C. Finally, the sample was extracted once with 1 vol. phenol/chloroform. After addition of sodium acetate, pH 5.5, the DNA was precipitated and resuspended in 20 ml of agarose gel loading buffer. DNA was fractionated by 1.2% agarose gel electrophoresis in Tris–acetate buffer (Ausubel et al., 1987) Nucleic acids were transferred to Hybond-N+ nylon membranes (Amersham, Arlington Heights, IL), which were subsequently hybridized with recombinant fulllength HBV DNA labeled to high specific activity (2–4× 108 cpm/mg) and exposed. As demon-
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strated in Fig. 1, all replicative forms of HBV DNA (RC, relaxed-circular DNA; DS, doublestranded linear DNA; SS, single-stranded DNA) were detected by Southern blot hybridization after transfection of HuH-7 (lane 1), HH29 (lane 3) or HHY41 cells (lane 5). Control transfections using vector only (lanes 2, 4 and 6) did not show HBV-specific signals. Quantitative analysis of replication showed similar levels of viral replication for the three cell lines investigated (data not shown). Analysis of cell culture supernatants (day five after transfection) for the presence of HBsAg and HBeAg was performed using the Abbott IMX system (Abbott, Abbott Park, IL). As demonstrated in Fig. 2, hepatocyte cell lines HH29 and HHY41 secreted viral antigens after transfection at high levels, comparable to the amount synthesized by HuH-7 cells. These experiments demonstrate that hepatocyte lines HH29 and HHY41 are capable of supporting HBV replication and synthesis as well as secretion of viral antigens. The differentiated characteristics of human hepatocyte lines HH29 and HHY41 suggested that,
Fig. 2. Viral antigen secretion into the cell culture supernatant after transfection of the cell lines HuH-7, HH29 and HHY41. HBsAg and HBeAg levels were determined by immunoassay. HBV-positive patient serum served as positive control. NC, negative control.
Fig. 1. Replication of HBV in the hepatoma cell line HuH-7 and the hepatocyte cell lines HH29 and HHY41 assessed by Southern blot hybridization. Cells were transiently transfected as described in the text. HBV-HTD, replication-competent head-to-tail-dimer of HBV; NC, negative control; RC, relaxedcircular HBV DNA; DS, double-stranded linear DNA; SS, single-stranded HBV DNA.
unlike previously established human hepatoma cell lines, they may be infected in vitro by HBV. For in vitro infection studies, cell lines HH29, HHY41 and HuH-7 (which can be infected in vitro only at very low and almost undetectable levels and was used here as negative control) were incubated at 37°C overnight in normal culture medium with HBV-positive patient serum (HBs-
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Ag positive, HBeAg positive, 3×1010 genome equivalents per ml; multiplicity of infection (MOI)=200) in the presence of 4% polyethylene glycol (PEG) 8000. For each cell line tested, negative control incubations were performed with HBV-negative human serum. After extensive washing with PBS to remove adherent virus from the surface of cells in culture (five separate washing steps with 10 ml each; 3 min incubation time each), cells were incubated for 2 min with 50 mM glycine at pH 2.2 to release residual virus bound to the cell surface (Lu et al., 1996). Cells were then cultured in normal culture medium until day 7. Aliquots of cell culture supernatants were taken before washing, 2 h after washing and daily from day 2–7. Sequential analysis of cell culture supernatants for synthesis of HBeAg demonstrated that, after removing HBeAg contained in the infectious inoculum, no de novo synthesis of viral antigen was detectable in all three cell lines tested (data not shown). Likewise, no viral transcripts were detected in cellular lysates analyzed on day 2 (data not shown). When cell lysates were analyzed on day 7 after inoculation with HBV for the presence of nucleocapsid-associated HBV DNA (see legend to Fig. 3 for details), HBV-specific signals were detected by Southern blot hybridization of extracts from HH29 and HHY41 cells (Fig. 3, lanes 4 and 6). By contrast, no HBV-specific signal was detected in extracts from HuH-7 cells (Fig. 3, lane 2). No single-stranded HBV DNA and no replicative intermediates were observed in all three cell lines. HH29- and HHY41associated HBV-specific signals were compared with a marker for HBV double-stranded linear DNA (Fig. 3, lane 1) and with the HBV-specific signal obtained when infectious virus from serum was processed analogous to cellular lysates (Fig. 3, lane 8). The direct comparison of HH29- and HHY41-associated signals with these markers revealed that the specific signals obtained from these cells most likely originated from the infectious inoculum HBV being bound to the cell surface. Our experiments show that the human hepatocyte lines HH29 and HHY41 support HBV replication and antigen synthesis as well as secretion.
The absence of viral transcripts and antigen synthesis after in vitro infection experiments suggests, however, that no productive in vitro infection could be established in these cell lines. These findings are consistent with the block of one or more crucial steps of the viral life cycle between attachment of virus to the cell and viral pregenome transcription in these cells. These steps include viral entry, genome release, transport to the nucleus, and genome repair. Furthermore, proteolytic events may be involved in the process of viral entry into host cells (Lu et al., 1996), as demonstrated by the V8-protease-mediated entry of HBV into HepG2 hepatoma cells in culture. HepG2 cells inoculated with intact virus did not
Fig. 3. Southern blot analysis of cellular lysates from the in vitro infection study. Detection of nucleocapsid-associated HBV DNA in cellular lysates from cell lines HH29 (lane 4) and HHY41 (lane 6). Cells were lysed in 50 mM Tris (pH 8.0)/1 mM EDTA/1% Nonidet P-40. The lysate was incubated with 20 U/ml of DNase I and 150 U/ml micrococcal nuclease, followed by incubation with 1 mg/ml proteinase K for 16 h at 37°C. After extraction with 1 vol. phenol/chloroform, the DNA released from nucleocapsids was precipitated, fractionated by agarose gel electrophoresis and transferred to Hybond-N + nylon membranes. HBV-specific nucleic acid was detected with a 32P-labeled full-length HBV DNA probe. Note that no nucleocapsid-associated HBV DNA is detected in extracts from HuH-7 cells (lane 2) and after incubation with control serum (lanes 3, 5 and 7). Lane 1, double-stranded linear HBV DNA marker; lane 8, HBV-specific signal associated with virus present in the infectious inoculum. RC, relaxed-circular HBV DNA; DS, double-stranded linear HBV DNA.
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contain detectable levels of intracellular HBV DNA at any time following infection, whereas HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection with V8-proteasetreated virus. Currently little is known about the hepatocyte receptor for HBV. In this regard, it is noteworthy that viral particles appear to adhere tightly to cell lines HH29 and HHY41 under conditions that remove HBV to undetectable levels from HuH-7 cells. HepG2 hepatoma cells which were demonstrated to be infectable only after V8 protease treatment of HBV were not used in the experiments reported here. However, the observation by Gerlich and coworkers (Lu et al., 1996) that binding between hepatitis B surface (HBs) particles and HepG2 cells occurred only after treatment of HBs particles with V8 protease suggests that untreated HBV particles will not adhere to HepG2 cells at significant levels under the harsh washing conditions used in the experiment described here. Several cellular proteins which bind to the envelope proteins of HBV through the pre-S or S domain have been described (Neurath et al., 1992; Pontisso et al., 1992; Hertogs et al., 1993; Mehdi et al., 1994; Budkowska et al., 1995), but it has been difficult to firmly establish that such proteins are components of the HBV receptor. Recently, human annexin V, a protein present on hepatocyte plasma membranes, has been demonstrated to exhibit strong and specific binding to small HBs protein and has been implicated in the penetration of HBV particles in an in vitro rat hepatoma cell system (Gong et al., 1996; De Meyer et al., 1997). At present, no information is available about the presence of human annexin V on plasma membranes of cell lines HH29 and HHY41. It will be informative to determine whether human annexin V is involved in the extraordinarily tight association of HBV to cell lines HH29 and HHY41 and whether additional cell surface components are involved in the attachment process. In summary, the liver cell lines HH29 and HHY41 support HBV replication and viral antigen synthesis. They cannot be infected by HBV in vitro, however, despite an exceptionally strong
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association of HBV with these cells in culture. The cell lines HH29 and HHY41 may be useful for the study of important aspects of the biology of HBV as well as for the molecular characterization of some putative components of the HBV receptor complex.
Acknowledgements We would like to thank Ruth Keist, Barbara Niedero¨st and Birgit Hockenjos for excellent technical assistance.
References Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 1987. In: Janssen, K. (Ed.), Current Protocols in Molecular Biology. Wiley, New York. Blum, H.E., Galun, E., Liang, T.J., v. Weizsa¨cker, F., Wands, J.R., 1991. Naturally occurring missense mutation in the polymerase gene terminating hepatitis B virus replication. J. Virol. 65 (4), 1836 – 1842. Budkowska, A., Bedossa, P., Groh, F., Louise, A., Pillot, J., 1995. Fibronectin of human liver sinusoids binds hepatitis B virus: identification by an anti-idiotypic antibody bearing the internal image of the pre-S2 domain. J. Virol. 69 (2), 840 – 848. Chen, C., Okayama, H., 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7 (8), 2745 – 2752. De Meyer, S., Gong, Z.J., Suwandhi, W., van Pelt, J., Soumillion, A., Yap, S.H., 1997. Organ and species specificity of hepatitis B virus (HBV) infection: a review of literature with a special reference to preferential attachment of HBV to human hepatocytes. J. Viral. Hepat. 4 (3), 145 – 153. Ganem, D., Varmus, H.E., 1987. The molecular biology of the hepatitis B viruses. Annu. Rev. Biochem. 56 (651), 651 – 693. Gong, Z.J., De Meyer, S., Van Pelt, J.F., Hertogs, K., Soumillion, A., Fevery, J., Yap, S.H., 1996. Human annexin V is the determining factor for cell infectability by hepatitis B virus. Hepatology 24 (4, Pt. 2), 248A. Hertogs, K., Leenders, W.P., Depla, E., De, B.W., Meheus, L., Raymackers, J., Moshage, H., Yap, S.H., 1993. Endonexin II, present on human liver plasma membranes, is a specific binding protein of small hepatitis B virus (HBV) envelope protein. Virology 197 (2), 549 – 557. Kono, Y., Yang, S., Letarte, M., Roberts, E.A., 1995. Establishment of a human hepatocyte line derived from primary culture in a collagen gel sandwich culture system. Exp. Cell Res. 221 (2), 478 – 485.
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Lu, X., Block, T.M., Gerlich, W.H., 1996. Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line. J. Virol. 70 (4), 2277–2285. Mabit, H., Dubanchet, S., Capel, F., Dauguet, C., Petit, M.A., 1994. In vitro infection of human hepatoma cells (HepG2) with hepatitis B virus (HBV): spontaneous selection of a stable HBV surface antigen-producing HepG2 cell line containing integrated HBV DNA sequences. J. Gen. Virol. 75 (Pt 10), 2681 – 2689. Mehdi, H., Kaplan, M.J., Anlar, F.Y., Yang, X., Bayer, R., Sutherland, K., Peeples, M.E., 1994. Hepatitis B virus surface antigen binds to apolipoprotein H. J. Virol. 68 (4), 2415 – 2424. Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T., Sato, J., 1982. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 42 (9), 3858–3863.
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Neurath, A.R., Strick, N., Li, Y.Y., 1992. Cells transfected with human interleukin 6 cDNA acquire binding sites for the hepatitis B virus envelope protein. J. Exp. Med. 176 (6), 1561 – 1569. Pontisso, P., Ruvoletto, M.G., Tiribelli, C., Gerlich, W.H., Ruol, A., Alberti, A., 1992. The preS1 domain of hepatitis B virus and IgA cross-react in their binding to the hepatocyte surface. J. Gen. Virol. 73 (8), 2041 – 2045. Roberts, E.A., Letarte, M., Squire, J., Yang, S., 1994. Characterization of human hepatocyte lines derived from normal liver tissue. Hepatology 19 (6), 1390 – 1399. Sells, M.A., Chen, M.L., Acs, G., 1987. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc. Natl. Acad. Sci. USA 84 (4), 1005 – 1009. Sleigh, M.J., 1986. A non-chromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells. Anal. Biochem. 156 (1), 251 – 256.
Hepatitis B virus replication and viral antigen synthesis in hepatocyte lines derived from normal human liver1 Jasper zu Putlitz 2,a, Eve A. Roberts b, Stefan Wieland a, Yumi Kono 3,b, Hubert E. Blum a,* a
Department of Internal Medicine II, Uni6ersity Hospital Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany b The Hospital for Sick Children Research Institute, Uni6ersity of Toronto, Toronto, Ont., Canada Received 9 May 1997; received in revised form 6 September 1997; accepted 15 September 1997
Abstract Transient transfection and in vitro infection experiments were performed to characterize replication and antigen synthesis of the hepatitis B virus (HBV) in human hepatocyte lines HH29 and HHY41, derived from normal liver tissue. These liver cell lines are capable of supporting HBV replication and gene expression at levels similar to the human hepatoma cell line HuH-7. Strikingly, a very tight adhesion of HBV to the outer cell membrane of HH29 and HHY41 was observed under conditions that removed HBV to undetectable levels from HuH-7 hepatoma cells. However, no productive HBV infection could be established in these cells as determined by the absence of viral transcripts and de novo antigen synthesis. In conclusion, the human hepatocyte cell lines HH29 and HHY41 may be useful to study important aspects of late steps in the replication of HBV, but appear to lack certain cellular components that play a pivotal role during early steps of the viral life cycle. © 1997 Elsevier Science B.V. Keywords: Hepatitis B virus; Hepatocytes; Transfection; Receptor
* Corresponding author. Tel.: +49 761 2703403; fax: + 49 761 2703610. 1 Presented in part at the 1996 Annual Meeting of the American Association for the Study of Liver Diseases and published in abstract form (Hepatology 1996;24(4):224A). 2 Present address: Molecular Hepatology Laboratory, MGH Cancer Center, Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA. 3 Present address: Department of Pediatrics, Faculty of Medicine, Tottori University, Yonago, Japan.
Chronic hepatitis B virus (HBV) infection is a major public health problem. Although the replication strategy of HBV is fairly well understood (Ganem and Varmus, 1987), little is known about the cellular receptor for HBV and early events in the viral life cycle, due to the lack of an efficient in vitro infection system. Previously established hepatoma cell lines, such as HuH-7 (Nakabayashi et al., 1982) and HepG2 (Sells et al., 1987), can be
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infected in vitro only at very low efficiency (Mabit et al., 1994), but support the replication of HBV when plasmid-encoded, replication-competent overlength constructs of HBV DNA are transfected into these cells. We investigated whether the human hepatocyte lines HH29 and HHY41 support the replication of HBV. Because these cells were derived from normal liver tissue rather than from a hepatocellular carcinoma, we reasoned that they might have retained cellular components that are essential during viral entry and/or early phases of the viral life cycle in the host cell. We therefore attempted to infect these cells in vitro with HBV. The human hepatocyte cell lines HH29 and HHY41 were previously established from surgically resected, normal liver tissue at The Hospital for Sick Children Research Institute, Toronto, Canada. HH29 was established by co-culture with rat liver epithelial cells (RLEC) as described (Roberts et al., 1994). HHY41 was established by prolonged culture between two layers of hydrated collagen (collagen gel sandwich) in a medium enriched with growth factors (Kono et al., 1995). These cell lines exhibit morphological and functional characteristics of differentiated hepatocytes. They retain glucose-6-phosphatase activity, produce albumin and other proteins typical of hepatocytes, and maintain various active cytochromes P-450. HH29 and HHY41 were grown in Dulbecco’s modification of Eagle’s medium (DMEM)/F12 (Life Technologies, Gaithersburg, MD) supplemented with 8% FCS, insulin 10 mg/ml, hydrocortisone 10 − 6 M, linoleic acid/bovine serum albumin 0.5 mg/ml/0.05 mg/ml, thyrotropin-releasing hormone 10 − 6 M, selenous acid 10 − 7 M (all supplements from Sigma, St. Louis, MO) and 1% penicillin/streptomycin stock solution (5000 U/ml penicillin G sodium/5000 mg/ml streptomycin). The cell line HuH-7 (Nakabayashi et al., 1982), derived from a human hepatocellular carcinoma, was grown in Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies), supplemented with 8% fetal calf serum (FCS), 1% non-essential amino acid solution (Life Technologies) and 1% penicillin/streptomycin stock solution (5000 U/ml penicillin G sodium/5000 mg/ml streptomycin; Life Technologies).
The human hepatocyte lines HH29 and HHY41 were transiently transfected using a modified calcium phosphate precipitation protocol (Chen and Okayama, 1987), routinely using 10 mg of a headto-tail dimer of wild-type HBV, subtype adw (ADW-HTD; GenBank™ accession number X02763; Blum et al., 1991) plus 1 mg of reporter plasmid (pCAT-Control; Promega, Madison, WI) per 100-mm plate. The variation in transfection efficiencies was assessed by CAT assay as described (Sleigh, 1986). The construct ADW-HTD allows for the replication of HBV. As a consequence of the synthesis of viral DNA and proteins, viral antigens are released into the cell culture supernatant, and nucleocapsid-associated replication products can be detected in cellular lysates. As positive control for virus replication, transfections were performed in parallel with the human hepatoma cell line HuH-7 which is known to support the replication of HBV. After incubation for 5 days, cellular lysates were analyzed for the presence of HBV nucleocapsid-associated nucleic acid. For the preparation of nucleocapsid-associated HBV DNA, transfected cells were lysed in 50 mM Tris (pH 8.0)/1 mM EDTA/1% Nonidet P-40. The lysate was centrifuged at 10 000× g for 5 min at room temperature. After the addition of CaCl2 and MgCl2 to a final concentration of 10 mM each, the supernatant was incubated with 20 U/ml of DNase I (Boehringer Mannheim) and micrococcal nuclease (final concentration 150 U/ ml; Pharmacia Biotech) for 2 h at 37°C. Next, EDTA (final concentration, 20 mmol/l), 10% sodium dodecyl sulfate (final concentration, 1%) and proteinase K (final concentration, 1 mg/ml; Promega) was added, followed by incubation for 12–16 h at 37°C. Finally, the sample was extracted once with 1 vol. phenol/chloroform. After addition of sodium acetate, pH 5.5, the DNA was precipitated and resuspended in 20 ml of agarose gel loading buffer. DNA was fractionated by 1.2% agarose gel electrophoresis in Tris–acetate buffer (Ausubel et al., 1987) Nucleic acids were transferred to Hybond-N+ nylon membranes (Amersham, Arlington Heights, IL), which were subsequently hybridized with recombinant fulllength HBV DNA labeled to high specific activity (2–4× 108 cpm/mg) and exposed. As demon-
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strated in Fig. 1, all replicative forms of HBV DNA (RC, relaxed-circular DNA; DS, doublestranded linear DNA; SS, single-stranded DNA) were detected by Southern blot hybridization after transfection of HuH-7 (lane 1), HH29 (lane 3) or HHY41 cells (lane 5). Control transfections using vector only (lanes 2, 4 and 6) did not show HBV-specific signals. Quantitative analysis of replication showed similar levels of viral replication for the three cell lines investigated (data not shown). Analysis of cell culture supernatants (day five after transfection) for the presence of HBsAg and HBeAg was performed using the Abbott IMX system (Abbott, Abbott Park, IL). As demonstrated in Fig. 2, hepatocyte cell lines HH29 and HHY41 secreted viral antigens after transfection at high levels, comparable to the amount synthesized by HuH-7 cells. These experiments demonstrate that hepatocyte lines HH29 and HHY41 are capable of supporting HBV replication and synthesis as well as secretion of viral antigens. The differentiated characteristics of human hepatocyte lines HH29 and HHY41 suggested that,
Fig. 2. Viral antigen secretion into the cell culture supernatant after transfection of the cell lines HuH-7, HH29 and HHY41. HBsAg and HBeAg levels were determined by immunoassay. HBV-positive patient serum served as positive control. NC, negative control.
Fig. 1. Replication of HBV in the hepatoma cell line HuH-7 and the hepatocyte cell lines HH29 and HHY41 assessed by Southern blot hybridization. Cells were transiently transfected as described in the text. HBV-HTD, replication-competent head-to-tail-dimer of HBV; NC, negative control; RC, relaxedcircular HBV DNA; DS, double-stranded linear DNA; SS, single-stranded HBV DNA.
unlike previously established human hepatoma cell lines, they may be infected in vitro by HBV. For in vitro infection studies, cell lines HH29, HHY41 and HuH-7 (which can be infected in vitro only at very low and almost undetectable levels and was used here as negative control) were incubated at 37°C overnight in normal culture medium with HBV-positive patient serum (HBs-
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Ag positive, HBeAg positive, 3×1010 genome equivalents per ml; multiplicity of infection (MOI)=200) in the presence of 4% polyethylene glycol (PEG) 8000. For each cell line tested, negative control incubations were performed with HBV-negative human serum. After extensive washing with PBS to remove adherent virus from the surface of cells in culture (five separate washing steps with 10 ml each; 3 min incubation time each), cells were incubated for 2 min with 50 mM glycine at pH 2.2 to release residual virus bound to the cell surface (Lu et al., 1996). Cells were then cultured in normal culture medium until day 7. Aliquots of cell culture supernatants were taken before washing, 2 h after washing and daily from day 2–7. Sequential analysis of cell culture supernatants for synthesis of HBeAg demonstrated that, after removing HBeAg contained in the infectious inoculum, no de novo synthesis of viral antigen was detectable in all three cell lines tested (data not shown). Likewise, no viral transcripts were detected in cellular lysates analyzed on day 2 (data not shown). When cell lysates were analyzed on day 7 after inoculation with HBV for the presence of nucleocapsid-associated HBV DNA (see legend to Fig. 3 for details), HBV-specific signals were detected by Southern blot hybridization of extracts from HH29 and HHY41 cells (Fig. 3, lanes 4 and 6). By contrast, no HBV-specific signal was detected in extracts from HuH-7 cells (Fig. 3, lane 2). No single-stranded HBV DNA and no replicative intermediates were observed in all three cell lines. HH29- and HHY41associated HBV-specific signals were compared with a marker for HBV double-stranded linear DNA (Fig. 3, lane 1) and with the HBV-specific signal obtained when infectious virus from serum was processed analogous to cellular lysates (Fig. 3, lane 8). The direct comparison of HH29- and HHY41-associated signals with these markers revealed that the specific signals obtained from these cells most likely originated from the infectious inoculum HBV being bound to the cell surface. Our experiments show that the human hepatocyte lines HH29 and HHY41 support HBV replication and antigen synthesis as well as secretion.
The absence of viral transcripts and antigen synthesis after in vitro infection experiments suggests, however, that no productive in vitro infection could be established in these cell lines. These findings are consistent with the block of one or more crucial steps of the viral life cycle between attachment of virus to the cell and viral pregenome transcription in these cells. These steps include viral entry, genome release, transport to the nucleus, and genome repair. Furthermore, proteolytic events may be involved in the process of viral entry into host cells (Lu et al., 1996), as demonstrated by the V8-protease-mediated entry of HBV into HepG2 hepatoma cells in culture. HepG2 cells inoculated with intact virus did not
Fig. 3. Southern blot analysis of cellular lysates from the in vitro infection study. Detection of nucleocapsid-associated HBV DNA in cellular lysates from cell lines HH29 (lane 4) and HHY41 (lane 6). Cells were lysed in 50 mM Tris (pH 8.0)/1 mM EDTA/1% Nonidet P-40. The lysate was incubated with 20 U/ml of DNase I and 150 U/ml micrococcal nuclease, followed by incubation with 1 mg/ml proteinase K for 16 h at 37°C. After extraction with 1 vol. phenol/chloroform, the DNA released from nucleocapsids was precipitated, fractionated by agarose gel electrophoresis and transferred to Hybond-N + nylon membranes. HBV-specific nucleic acid was detected with a 32P-labeled full-length HBV DNA probe. Note that no nucleocapsid-associated HBV DNA is detected in extracts from HuH-7 cells (lane 2) and after incubation with control serum (lanes 3, 5 and 7). Lane 1, double-stranded linear HBV DNA marker; lane 8, HBV-specific signal associated with virus present in the infectious inoculum. RC, relaxed-circular HBV DNA; DS, double-stranded linear HBV DNA.
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contain detectable levels of intracellular HBV DNA at any time following infection, whereas HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection with V8-proteasetreated virus. Currently little is known about the hepatocyte receptor for HBV. In this regard, it is noteworthy that viral particles appear to adhere tightly to cell lines HH29 and HHY41 under conditions that remove HBV to undetectable levels from HuH-7 cells. HepG2 hepatoma cells which were demonstrated to be infectable only after V8 protease treatment of HBV were not used in the experiments reported here. However, the observation by Gerlich and coworkers (Lu et al., 1996) that binding between hepatitis B surface (HBs) particles and HepG2 cells occurred only after treatment of HBs particles with V8 protease suggests that untreated HBV particles will not adhere to HepG2 cells at significant levels under the harsh washing conditions used in the experiment described here. Several cellular proteins which bind to the envelope proteins of HBV through the pre-S or S domain have been described (Neurath et al., 1992; Pontisso et al., 1992; Hertogs et al., 1993; Mehdi et al., 1994; Budkowska et al., 1995), but it has been difficult to firmly establish that such proteins are components of the HBV receptor. Recently, human annexin V, a protein present on hepatocyte plasma membranes, has been demonstrated to exhibit strong and specific binding to small HBs protein and has been implicated in the penetration of HBV particles in an in vitro rat hepatoma cell system (Gong et al., 1996; De Meyer et al., 1997). At present, no information is available about the presence of human annexin V on plasma membranes of cell lines HH29 and HHY41. It will be informative to determine whether human annexin V is involved in the extraordinarily tight association of HBV to cell lines HH29 and HHY41 and whether additional cell surface components are involved in the attachment process. In summary, the liver cell lines HH29 and HHY41 support HBV replication and viral antigen synthesis. They cannot be infected by HBV in vitro, however, despite an exceptionally strong
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association of HBV with these cells in culture. The cell lines HH29 and HHY41 may be useful for the study of important aspects of the biology of HBV as well as for the molecular characterization of some putative components of the HBV receptor complex.
Acknowledgements We would like to thank Ruth Keist, Barbara Niedero¨st and Birgit Hockenjos for excellent technical assistance.
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