Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA.

Tsurimoto, Toshiki; Fujiyama, Asao; Matsubara, Kazuo

doi:10.1073/pnas.84.2.444

Cited by 182 publications

(125 citation statements)

References 28 publications

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“…5 Within the last decade, several HBV expressing cell lines have been established by transfecting viral DNA into liverderived human cell lines and by selecting novel cell lines containing stably integrated HBV genomes. [6][7][8] The most widely used are the HepG2 2.2.15 cell line (2.2.15) derived from the HepG2 hepatoblastoma cell line and HB611 derived from the HuH6 hepatoma cell line. These and other cell lines have led to considerable progress in the study of HBV in vitro.…”

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confidence: 99%

Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus

1998

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Hepatitis B virus (HBV) is a small, double-stranded DNA virus and is the prototype of the hepadnavirus family. HBV is a human pathogen capable of causing both acute and chronic hepatitis. The World Health Organization currently estimates that 350 million people are chronically infected with HBV. Persistent HBV infection is also associated with an increased risk of cirrhosis and hepatocellular carcinoma. 1 Although a tremendous amount is known about HBV, our knowledge of the virus is by no means complete. Historically, major obstacles in the study of HBV have been the inability of the virus to infect cells in vitro, and the lack of animal model systems due to a strict virus-host range. Thus, many aspects of HBV biology have been unraveled by studying related hepadnaviruses, such as the duck hepatitis virus which is capable of in vitro infection, 2 and the woodchuck hepatitis virus which allows for the in vivo study in an animal model system. 3 The duck hepatitis virus and woodchuck hepatitis virus systems were instrumental in developing an understanding of the hepadnavirus lifecycle and remain valuable models for HBV infection. However, many significant differences exist between animal hepadnaviruses and HBV. For example, avian hepatitis viruses do not encode the X gene, 4 and major transcriptional differences between woodchuck hepatitis virus and HBV have been reported. 5 Within the last decade, several HBV expressing cell lines have been established by transfecting viral DNA into liverderived human cell lines and by selecting novel cell lines containing stably integrated HBV genomes. [6][7][8] The most widely used are the HepG2 2.2.15 cell line (2.2.15) derived from the HepG2 hepatoblastoma cell line and HB611 derived from the HuH6 hepatoma cell line. These and other cell lines have led to considerable progress in the study of HBV in vitro. However, there are some inherent drawbacks which preclude the use of these cell lines in studying some aspects of HBV biology. (1) Many HBV expressing cell lines were created using constructs containing strong heterologous promoters proximal to the HBV genome. The effect those promoters have on HBV transcription and replication is unclear but could differ substantially from what occurs in a natural infection in vivo in which HBV gene expression is driven solely by endogenous HBV promoters. (2) Cell lines commonly used to study HBV contain multiple copies of integrated HBV DNA. Unlike retroviruses, which integrate viral DNA into the host genome, hepadnavirus genomes are not integrated routinely but, instead, are maintained in the nucleus of infected cells in vivo as a pool of episomal covalently closed circular (CCC) DNA molecules. 9 Although the integration of HBV DNA in human liver has been reported, 10 it is not an obligatory part of the HBV lifecycle. HBV does not encode any machinery for integration into the host genome, and integration is not required for HBV replication. In addition, when integrated HBV DNA is found,

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confidence: 99%

Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus

1998

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“…By the time the studies described above were completed, most work on hepadnavirus replication was shifting away from animal models to transfection-based studies using liver tumor cell lines (Tsurimoto et al 1987;Yaginuma et al 1987;Condreay et al 1990). Further work with animal models focused primarily on understanding the host response to infection and development of antiviral therapies.…”

Section: The Hepadnavirus Life Cyclementioning

confidence: 99%

“…It was not until 1986 that the HepG2 line of human liver tumor cells (Aden et al 1979) was found to support HBV replication from cloned viral DNA (Sureau et al 1986;Sells et al 1987). In the following 3 years, two other human liver tumor lines, Huh6 and Huh7, and a rat hepatoma cell line were also shown to fulfill this need (Tsurimoto et al 1987;Yaginuma et al 1987;Shih et al 1989). As discussed below, by this time many key steps in hepadnavirus replication had been revealed through the use of animal models and primary hepatocyte cultures.…”

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Animal Models and the Molecular Biology of Hepadnavirus Infection

Mason

2015

Cold Spring Harbor Perspectives in Medicine

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Australian antigen, the envelope protein of hepatitis B virus (HBV), was discovered in 1967 as a prevalent serum antigen in hepatitis B patients. Early electron microscopy (EM) studies showed that this antigen was present in 22-nm particles in patient sera, which were believed to be incomplete virus. Complete virus, much less abundant than the 22-nm particles, was finally visualized in 1970. HBV was soon found to infect chimpanzees, gorillas, orangutans, gibbon apes, and, more recently, tree shrews (Tupaia belangeri) and cynomolgus macaques (Macaca fascicularis). This restricted host range placed limits on the kinds of studies that might be performed to better understand the biology and molecular biology of HBV and to develop antiviral therapies to treat chronic infections. About 10 years after the discovery of HBV, this problem was bypassed with the discovery of viruses related to HBV in woodchucks, ground squirrels, and ducks. Although unlikely animal models, their use revealed the key steps in hepadnavirus replication and in the host response to infection, including the fact that the viral nuclear episome is the ultimate target for immune clearance of transient infections and antiviral therapy of chronic infections. Studies with these and other animal models have also suggested interesting clues into the link between chronic HBV infection and hepatocellular carcinoma.

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“…However, the differentiated state of the hepatocytes in these cultures can be maintained only for a limited amount of time. Propagation of HBV in cell culture has been achieved by transfection of closed circular HBV DNA in human hepatoma cell lines (HepG2 or HUH7) (Sureau et al, 1986;Sells et al, 1987;Tsurimoto et al, 1987). However, the latter system did not mimic the in vivo process of viral infection, including virus penetration and early biochemical events.…”

Section: Introductionmentioning

confidence: 99%

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

Mabit

Dubanchet

Capel

et al. 1994

Journal of General Virology

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The degree of susceptibility of human hepatoma (HepG2) cells to direct hepatitis B virus (HBV) infection remains unknown. We previously observed a low level of Dane particle production and viral DNA replication after in vitro infection of HepG2 cells with serum-derived HBV. However, this culture system appeared to be affected by variations as human hepatocyte cultures. In the present study, HBV infection of HepG2 cells led to a significant increase in the secretion of three envelope antigens (HBsAg, preS2Ag and preS1Ag) at 4 days post-infection, and Northern blot analysis revealed the presence of both preS1 (2.6 kb) and preS2/S (2.2 kb) transcripts. Expression of preS1Ag and the corresponding viral RNA became undetectable on 21 days post-infection whereas the 2"2 kb RNA species persisted and was associated with secretion of subviral HBs particles expressing preS2-epitopes and banding between 30 and 35% sucrose. At 35 days post-infection (fifth passage), a sudden high level production of HBsAg and preS1Ag was observed, followed by a massive cell death (90 %). A stable HBsAg-producing HepG2 cell line, designated HepG2-BV3, grew out of the surviving cells. HepG2-BV3 cells could integrate HBV DNA sequences and produce the three HBV surface antigens. Treatment with dexamethasone increased the HBsAg and preS1Ag secretion. Such a HBsAg-producing HepG2 cell line obtained by in vitro HBV infection seems to mimick events that occur in the naturally occurring persistent chronic infection, and therefore may be an efficient in vitro model for studying the contribution of viral integration in the dysregulation of HBV and liverspecific genes expression.

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Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA.

Cited by 182 publications

References 28 publications

Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus

Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus

Animal Models and the Molecular Biology of Hepadnavirus Infection

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

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