Evidence for Non-chromosomal Hepatitis B Virus Surface (HBsAg)- and Core Antigen (HBcAg)-specific DNA Sequences in a Hepatoma Cell Line

Marquardt, Otfried; Zaslavsky, V.; Hofschneider, Peter Hans

doi:10.1099/0022-1317-61-1-105

Cited by 9 publications

(4 citation statements)

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“…To establish the sensitivity of the in situ hybridization for nucleotide sequences, we hybridized a probe of cloned HBV DNA to human hepatoma cells (Alexander cells; PLC/PRF/5; refs. 27 and 28) that contain extrachromosomal (29) and integrated HBV DNA (13,(30)(31)(32)(33)(34) or to mouse L cells that lack HBV DNA. After radioautographic exposure for 8 weeks, the Alexander cells showed 12 grains per cell with a relatively uniform and predominantly nuclear distribution (Fig.…”

mentioning

confidence: 99%

Detection of hepatitis B virus DNA in hepatocytes, bile duct epithelium, and vascular elements by in situ hybridization.

Blum¹,

Stowring²,

A³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

143

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A radiolabeled probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue from three patients with chronic hepatitis B. The HBV genome was detected not only in infected hepatocytes but also in bile duct epithelial cells, endothelial cells, and smooth muscle cells. These findings extend the known host cell range for HBV, suggest new mechanisms of viral dissemination, and illustrate the usefiuness of in situ hybridization in the study of pathogenesis of HBV infection.HBV infection is a major public health problem, with an estimated 200 million persistently infected people worldwide (1)(2)(3). HBV is a DNA virus that infects only humans and certain nonhuman primates and causes a wide spectrum of acute and chronic liver disease (4). Recent evidence further suggests that this virus is involved in the etiology of hepatocellular carcinoma (4-13). In infected individuals, the virion and DNA-free particles of hepatitis B surface antigen (HBsAg) circulate in blood, and viral antigen has been found in most body fluids, including bile (4, 14-16). However, the study of the pathobiology of HBV infection is impeded by the current inability to propagate HBV in cell culture. The availability of cloned HBV DNA now permits us to investigate by in situ hybridization the presence and quantity of viral copies at the cellular level and their correlation with histologic lesions. This paper presents findings indicating that HBV DNA in virus-infected liver is detectable not only in hepatocytes, as previously reported by Gowans et al. (17), but also in bile duct epithelium and vascular elements, thereby identifying new and unsuspected target cells for HBV. Cellular DNA Extraction. PLC/PRF/5 cells in a confluent monolayer were trypsinized and washed once with phosphatebuffered saline. The cells were lysed at room temperature by incubation in 10 mM Tris HCI, pH 7.4/1 mM EDTA/100 mM NaCl/1% sodium dodecyl sulfate. After addition of proteinase K (Boehringer Mannheim) to a final concentration of 100 ,g/ ml, the lysate was incubated at 37°C for 12-16 hr. The solution was then extracted twice with buffer-saturated phenol/chloroform/isoamyl alcohol, 25:24:1 (vol/vol), and once with chloroform/isoamyl alcohol, 24:1. The nucleic acids were precipitated with 2 vol of ethanol. The precipitate was dissolved in 10 mM Tris'HCl, pH 7.4/1 mM EDTA/100 mM NaCl, treated with DNase-free RNase (Millipore) at 100 ,ug/ml for 3 hr at 370C, and extracted and precipitated as described above. The number of HBV DNA copies per Alexander cell was determined by dot blot analysis (22) with 32P-labeled HBV DNA as a probe (specific activity, 1-2 x 109 cpm/,ug) and a known amount of cloned HBV DNA or of DNA extracted from PLC/ PRF/5 cells. The hybridization was quantitated by liquid scintillation counting. The number of four copies of HBV DNA per Alexander cell was calculated by assuming a cellular DNA content of 6 pg. MATERIALS AND METHODSIn 6685The publication costs of this article were defrayed in part by page charge ...

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mentioning

confidence: 99%

Detection of hepatitis B virus DNA in hepatocytes, bile duct epithelium, and vascular elements by in situ hybridization.

Blum¹,

Stowring²,

A³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

143

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“…In addition to HBV DNA which is integrated into chromosomes nonintegrated HBV-specific DNA exists in PLC/PRF/5 cells according to publications from one laboratory (25,36,65). This DNA is found in the cell cytoplasm as a DNA-protein complex which exhibits a buoyant density of 1.3 g/ ml in CsC1.…”

Section: Extrachromosomal Hbv Dna In Plc/prf/5 Cellsmentioning

confidence: 99%

Hepatitis B virus components produced by the human hepatoma cell line PLC/PRF/5: Do they indicate virus propagation?

Marquardt

1986

Archives of Virology

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Today, hepatitis B is geographically one of the most wide-spread of human virus epidemics. The vL-us (HBV) is transmitted by close contact and causes a variety of liver diseases, ranging from inapparent forms to acute or ful~finant hepatitis, eba-onie hepatitis and liver cirrhosis (reviewed in 56). HBV is found further in lymphoblastoid (13, 49) and pancreatic (24) cells. Perinatal HBV infection causes chronic hepatitis in more than 90 per cent of the cases (4). Moreover, those patients have approximately a 200-fold excess risk to develop hepatoeellular carcinoma (5) so that HBV is considered a causative agent for liver cancer in man. However, our understanding of HBV biology is limited, for attempts to multiply HBV in convenient experimental systems have been unsuccessful. The Virus-How to Study Replication?The host range of HBV is apparently narrow. Only HBV-infected chimpanzees (reviewed in 15) and infected patients allow studies on HBV replication. Evidence thus has been obtained for assembly of HBV in the cy%o-plasm of cells (6) and passage through cell membrane without necessarily causing cell lysis (58). However, such investigations are expensive and time consuming, facts which induced the search for alternative approaches.One approach makes use of the HBV-related viruses ~(HV, GSHV and DHBV which infect woodchucks (55), Beechey ground squirrels (35) and 2 0. MARQUARDT Pekin ducks (40). In the DHBV system, the virus replicates via an RNA intermediate. This has recently been shown to be also the case with HBV (42). On the other hand, DHBV differs from HBV in genome organization. Furthermore, hepatocellular carcinoma has not been observed in Pekin ducks and ground squirrels. This limits the model character of these systems for HBV research.Another apprach to understand HBV replication is based on the cultivation of cells positive for HBV components. The propagation of differentiated HBV-infected hepatocytes or pancreatic cells in vitro proved to be troublesome. The interest therefore focussed on persistently HBV-infected hepatoma cell lines derived from patients (1, 3, 27) and on cell lines which have been transfected in vitro with HBV DNA (12,20,21,23,62). Whether infectious virus particles are produced by such cells remains to be demonstrated despite the detection of several virus components. The approach may nevertheless be successful since chimpanzees develop hepatitis B when their hepatocytes are inoculated with cloned HBV DNA (63). The most detailed studies of naturally persistent HBV infection have been done on the hepatoma cell line PLC/PRF/5 (3). The object of this review is to summarize the data obtained so far with this cell line and to correlate them with the virus structure and genome organization. An evaluation of the system for the understanding of HBV replication may be both possible and useful at this point.

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“…One of them, the human hepatoma cell line PLC/PRF/5 (Macnab et al, 1976), contains HBV DNA specific for the surface antigen (HBsAg) and the core antigen (HBcAg) both extrachromosomally (Zaslavsky et al, 1980;Marquardt et al, 1982) and integrated into chromosomes (Edman et al, 1980;Miller & Robinson, 1983). HBsAg is produced by the cells and functionally active genes were found among the HBV DNA copies integrated into the chromosomes (Dejean et al, 1983;Koshy et al, 1983).…”

mentioning

confidence: 99%

“…The homogenization was controlled by microscopic observation. Culture of the cells in vitro was described previously (Marquardt et al, 1982) and ceils were homogenized as described above. Homogenized cells were investigated for the presence of HBsAg and HBc/HBeAg using radioimmunoassays (RIA).…”

mentioning

confidence: 99%

Expression of Hepatitis B Virus Core Antigen Gene Is Induced in Human Hepatoma Cells by Their Growth in Nude Mice

Marquardt

Loringhoven

Frösner

1984

Journal of General Virology

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Evidence for Non-chromosomal Hepatitis B Virus Surface (HBsAg)- and Core Antigen (HBcAg)-specific DNA Sequences in a Hepatoma Cell Line

Cited by 9 publications

References 10 publications

Detection of hepatitis B virus DNA in hepatocytes, bile duct epithelium, and vascular elements by in situ hybridization.

Detection of hepatitis B virus DNA in hepatocytes, bile duct epithelium, and vascular elements by in situ hybridization.

Hepatitis B virus components produced by the human hepatoma cell line PLC/PRF/5: Do they indicate virus propagation?

Expression of Hepatitis B Virus Core Antigen Gene Is Induced in Human Hepatoma Cells by Their Growth in Nude Mice

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