In vitro experimental infection of primary human hepatocytes with hepatitis B virus

Galle, Peter R.; Hagelstein, Jens; Kommerell, B; Volkmann, Martin; Schranz, Peter; Zentgraf, Hanswalter

doi:10.1016/0016-5085(94)90700-5

Cited by 118 publications

(90 citation statements)

References 30 publications

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“…5). The efficiency of HBV infection of primary tupaia hepatocytes in vitro, based on the level of HBsAg and HBeAg secreted into culture medium, is comparable with the infection of primary human hepatocytes 28,29 and the human hepatoma cell line HepG2. 30,31 Without in situ hybridization data it is impossible, however, to define infection efficiency more precisely.…”

Section: Resultsmentioning

confidence: 77%

Hepatitis B Virus Infection of Tupaia Hepatocytes In Vitro And In Vivo

et al. 1996

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treatment of tree shrews in China 15,16 as well For the systematic analysis of various clinical and moas the easy adaptation of these animals to the laboratory lecular aspects of hepatitis B virus (HBV) infection, an environment, 17,18 we systematically analyzed the tree shrew experimental small animal system of HBV infection species tupaia belangeri for the study of HBV infection in would be a great advance. The susceptibility to HBV invitro and in vivo. fection, therefore, of hepatocytes from the tree shrew species tupaia belangeri was studied in vitro and in vivo. MATERIALS AND METHODS Primary hepatocytes isolated from livers of tupaias can be reproducibly infected with HBV. In vitro infectionTupaia Colony. For breeding purposes, animals (tupaia belangeri) were obtained from the German Primate Center, Göttingen, Gerresults in viral DNA and RNA synthesis in hepatocytes many. They were bred and maintained at the animal facilities of and secretion hepatitis B surface antigen (HBsAg) and the University Hospital Zü rich in accordance with institutionally hepatitis B e antigen (HBeAg) into culture medium. Tuapproved protocols. Under optimal conditions (room temperature, paias can also be infected with HBV in vivo, resulting 28ЊC; relative humidity, 70%; day-night light cycle, 12 and 12 hours), in viral DNA replication and gene expression in tupaia the mothers give birth to two to three animals every 8 to 10 weeks. livers. Similar to acute, self-limited hepatitis B in hu-At birth, the animals are about 6 to 7 cm long (without tail) and mans, HBsAg is rapidly cleared from serum, followed weigh 16 to 19 g. Within 10 to 12 weeks, they reach adulthood with by seroconversion to anti-HBe and anti-HBs. These data an average length of 12 to 18 cm (without tail) and 180 to 200 g body weight. All animals were negative for serological markers of HBV clearly show that HBV is infectious to tupaia hepato- problem worldwide and is associated with a wide spectrum of exclusion, was 90% to 95%. The cells were washed and seeded at a clinical presentations, ranging from the asymptomatic HBV density of 3 1 10 6 viable cells/10 mL medium on collagen-coated 100-carrier status to the development of cirrhosis and hepatocel-mm tissue culture plates. Plating efficiency was 85%. Cells were lular carcinoma. 1 HBV represents the prototypic member of maintained in Williams' E medium with Glutamax J, buffered with the hepadnaviruses 2,3 and many molecular and clinical as-15 mmol/L hydroxyethyl piperazine ethane sulfuric acid (Sigma), pH 7.4, and supplemented with 1.5% dimethyl sulfoxide (HybriMax, pects of HBV infection have been defined. 4,5 HBV naturally Sigma), 0.1 mmol/L insulin, 0.1 mmol/L dexamethasone, 100 mg/mL infects only humans and experimentally chimpanzees. While penicillin, and 100 mg/mL streptomycine.various aspects of the pathogenesis of HBV-related liver disTransient Transfection of Primary Tupaia Hepatocytes. Transfecease can be studied in transgenic animals 6-9 and animal modtion of primary tupaia hepatocytes with a replicatio...

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Section: Resultsmentioning

confidence: 77%

Hepatitis B Virus Infection of Tupaia Hepatocytes In Vitro And In Vivo

et al. 1996

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“…The primary human liver cells were freshly isolated from different human livers. The resistance of these hepatocytes to H1 virus ± induced cell killing cannot simply be attributed to their nondividing status because they can traverse the S -phase under the culture conditions used, 30,36,38 which is a prerequisite for H1 to replicate and to become cytocidal. 13 It may be argued that long -time culturing of the tumor-derived cells may have caused a drift to a greater permissivity of those tumor cells to parvovirus infection.…”

Section: Discussionmentioning

confidence: 99%

“…29 The simian virus 40 ± transformed newborn human kidney cell line (NB -E ) was propagated in minimal essential medium (Life Technologies ) with 5% fetal calf serum, 5 mmol / L glutamine, and 100 g /mL penicillin. 22 Exponentially growing cultures 30 Briefly, the resected piece of liver was perfused via a central vessel, first with Dulbecco's modified Eagle's medium, and then with William's medium E (WME ) containing 0.05% collagenase and 5 mmol /L calcium. Cell suspension was filtered through a gauze, washed four times with ice -cold WME containing 5% fetal calf serum, and seeded at a density of 1Â10 5 viable cells/cm 2 on collagen -coated culture dishes.…”

Section: Tumor Cells and Primary Human Hepatocytesmentioning

confidence: 99%

“…For cell maintenance, WME was used, supplemented with 0.066 mol /L insulin, 0.1 mol / L glucagon, 0.1 mol /L triiodothyronine, 5 mmol /L glutamine, 37 mol / L inosine, 10 g /mL gentamycin, 100 g/ mL penicillin, and 20 mmol /L Hepes. 30 Virus infection, production, and cytotoxicity H1 virus was propagated in NB -E cells and purified over cesium chloride gradients as described earlier. 22 Wildtype (wt ) H1 titration by plaque assay was performed according to published methods.…”

Section: Tumor Cells and Primary Human Hepatocytesmentioning

confidence: 99%

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Effective infection, apoptotic cell killing and gene transfer of human hepatoma cells but not primary hepatocytes by parvovirus H1 and derived vectors

Moehler¹,

Blechacz

Weiskopf³

et al. 2001

Cancer Gene Ther

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Autonomous parvoviruses preferentially replicate in and kill in vitro ± transformed cells and reduce the incidence of spontaneous and implanted tumors in animals. Because of these natural oncotropic and oncolytic properties, parvoviruses deserve to be considered as potential antitumor vectors. Here, we assessed whether parvovirus H1 is able to kill human hepatoma cells by induction of apoptosis but spares primary human liver cells, and whether the former cells can efficiently be transduced by H1 virus ± based vectors. Cell death, infectivity, and transgene transduction were investigated in Hep3B, HepG2, and Huh7 cells and in primary human hepatocytes with natural and recombinant H1 virus. All hepatoma cells were susceptible to H1 virus ± induced cytolyis. Cell death correlated with H1 virus DNA replication, nonstructural protein expression, and with morphological features of apoptosis. H1 virus ± induced apoptosis was more pronounced in p53 -deleted Hep3B and p53 -mutated Huh7 cells than in HepG2 cells which express wild -type p53. In Hep3B cells, apoptosis was partially inhibited by DEVD -CHO, a caspase -3 inhibitor. In contrast, H1 virus ± infected primary hepatocytes were neither positive for nonstructural protein expression nor susceptible to H1 virus ± induced killing. Infection with a recombinant parvovirus vector carrying the luciferase gene under control of parvovirus promoter P38 led to higher transgene activities in hepatoma cells than in the hepatocytes. Taken together, H1 virus kills human hepatoma cells at low virus multiplicity but not primary hepatocytes. Thus, recombinant H1 viruses carrying antitumor transgenes may be considered as potential therapeutic options for the treatment of hepatocellular carcinomas. Cancer Gene Therapy ( 2001 ) 8, 158 ± 167

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“…It has been postulated that HepG2 cells are not susceptible to HBV infection because they lack a fusion-activating protease for cleavage of L/M protein on the cell surface (40). That primary hepatocytes can be infected by HBV in vitro (55) suggests that this "V8 proteaselike activity" might be present on the surface of primary hepatocytes and that it is required for the natural course of HBV infection. Our data further indicate that this V8 protease-like activity is present in HuH-7 and HepG2 cells, as evidenced by mutation of aa 57, which rendered M protein unable to transactivate the S promoter and inactivated its nuclear translocation ability.…”

Section: Protein Regulates Surface Gene Expression By Interacting Wmentioning

confidence: 99%

Novel Autoregulatory Function of Hepatitis B Virus M Protein on Surface Gene Expression

Huang

Tsai

et al. 2005

Journal of Biological Chemistry

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The hepatitis B virus surface gene consists of a single open reading frame divided into three coding regions: pre-S1, pre-S2, and S. By alternate translation at each of the three initiation codons, L, M, and S proteins can be synthesized. Studies have shown that M protein is not essential for viral replication, virion morphogenesis, or in vitro infectivity. In this study, we show that native M protein can regulate surface gene expression at the transcriptional level. The regulatory effect of M protein is mediated through the CCAAT box within the S promoter. Deletion mapping analysis indicated that the transactivating effect of M protein is mediated through amino acids 1-57 of M protein (the MHBs au domain), although its maximal transactivation activity coincides with that of the pre-S2 domain. This conclusion is supported by the fact that disruption of the putative V8 protease site at the pre-S2/S domain junction not only rendered M protein incapable of transactivating the S promoter but also inactivated its nuclear translocation potential. Immunoprecipitation and immunoblot experiments demonstrated that pre-S2 interacts with the three subunits of the CCAAT box-binding factor/nuclear factor Y, the cognate binding protein of the CCAAT box. These results demonstrate and define a novel regulatory role of M protein, which, under natural conditions, may undergo a proteolytic process to generate an MHBs au species that will be translocated inside the nucleus, where it will interact with the CCAAT box-binding factor to regulate surface gene expression. Because the CCAAT box is located at a fixed position within numerous promoters, these observations might provide a plausible explanation for hepatitis B virus-associated hepatocarcinogenesis. Hepatitis B virus (HBV)1 infection causes a wide spectrum of liver diseases, including acute hepatitis, chronic active hepatitis, cirrhosis, and hepatocellular carcinoma (1-4). HBV belongs to the hepadnavirus family, and its particle coat consists of a lipid bilayer membrane and envelope proteins. It has a partial duplex DNA genome of ϳ3.2 kb and contains four overlapping open reading frames: DNA polymerase, core protein, surface antigen, and the X gene (5). The HBV surface (envelope) gene encodes three forms of viral surface proteins (6, 7). It is divided into three parts by two internal initiation codons: the pre-S1, pre-S2, and S regions. This combination of three regions forms the large surface antigen LHBsAg or L protein. In addition, the pre-S2 and S regions form the middle surface antigen MHBsAg or M protein. The small surface antigen SHBsAg or S protein, also called major S protein, constitutes the most abundant protein of HBV envelopes. The production of these three forms of surface protein (L, M, and S) is controlled by two tandem promoters. The upstream pre-S1 promoter controls the transcription of a 2.4-kb transcript that encodes L protein only. The downstream S promoter, ϳ240 bp away, specifies transcripts with heterogeneous 5Ј termini (5), with the largest transcript e...

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In vitro experimental infection of primary human hepatocytes with hepatitis B virus

Cited by 118 publications

References 30 publications

Hepatitis B Virus Infection of Tupaia Hepatocytes In Vitro And In Vivo

Hepatitis B Virus Infection of Tupaia Hepatocytes In Vitro And In Vivo

Effective infection, apoptotic cell killing and gene transfer of human hepatoma cells but not primary hepatocytes by parvovirus H1 and derived vectors

Novel Autoregulatory Function of Hepatitis B Virus M Protein on Surface Gene Expression

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