Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells

Hantz, Olivier; Parent, Romain; Durantel, David; Gripon, Philippe; Guguen‐Guillouzo, Christiane; Zoulim, Fabien

doi:10.1099/vir.0.004861-0

Cited by 129 publications

(134 citation statements)

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“…First, DHBV uses the L protein to regulate the amplification of cccDNA (30,31,49,50). Second, measurement of the level of cccDNA in HBV-infected patients, as well as experimental systems for the expression of HBV, indicates this molecule is present at ϳ1 to 10 copies per cell (2,4,10,15,18,20,28,(53)(54)(55), suggesting that its synthesis is regulated. Third, some studies observed changes in the level of cccDNA in response to ablating or restoring expression of the envelope proteins (15,18).…”

Section: Discussionmentioning

confidence: 99%

Roles of the Envelope Proteins in the Amplification of Covalently Closed Circular DNA and Completion of Synthesis of the Plus-Strand DNA in Hepatitis B Virus

Lentz

Loeb

2011

J Virol

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Covalently closed circular DNA (cccDNA), the nuclear form of hepatitis B virus (HBV), is synthesized by repair of the relaxed circular (RC) DNA genome. Initially, cccDNA is derived from RC DNA from the infecting virion, but additional copies of cccDNA are derived from newly synthesized RC DNA molecules in a process termed intracellular amplification. It has been shown that the large viral envelope protein limits the intracellular amplification of cccDNA for duck hepatitis B virus. The role of the envelope proteins in regulating the amplification of cccDNA in HBV is not well characterized. The present report demonstrates regulation of synthesis of cccDNA by the envelope proteins of HBV. Ablation of expression of the envelope proteins led to an increase (>6-fold) in the level of cccDNA. Subsequent restoration of envelope protein expression led to a decrease (>50%) in the level of cccDNA, which inversely correlated with the level of the envelope proteins. We found that the expression of L protein alone or in combination with M and/or S proteins led to a decrease in cccDNA levels, indicating that L contributes to the regulation of cccDNA. Coexpression of L and M led to greater regulation than either L alone or L and S. Coexpression of all three envelope proteins was also found to limit completion of plus-strand DNA synthesis, and the degree of this effect correlated with the level of the proteins and virion secretion.Hepatitis B virus (HBV) is a member of the family Hepadnaviridae. Features of this family include a small, doublestranded DNA genome and enveloped virions (14). The envelope of HBV consists of a host cell-derived phospholipid bilayer that contains the virally encoded large (L), middle (M), and small (S) envelope proteins (21). This envelope surrounds the nucleocapsid, which is composed of the viral core (C) protein and contains the DNA genome (11). The doublestranded DNA genome within the virion is in either a relaxed circular (RC) or a duplex linear (DL) conformation, of which RC is the predominant form (12,44,48). RC DNA contains a full-length minus strand, which has the viral polymerase (P) protein covalently attached to its 5Ј end (17). The plus strand has a specific 5Ј end but is extended to various lengths, which can leave a single-stranded (SS) region (12,45,48). The reason the plus strand is not completely synthesized prior to the release of the virion is not understood. It is not known if the process of the nucleocapsid acquiring an envelope is related to the incomplete state of the plus strand found in virions.The RC DNA molecule from the virion is converted into a covalently closed circular DNA (cccDNA) molecule in the nucleus of a cell (38,40,51). The mechanism of conversion of RC DNA into cccDNA is not well understood. Minimally, this involves removal of the 5Ј-terminal moieties from the minus strand (P protein) and the plus strand (RNA primer), completion of synthesis of the plus strand, and ligation of 5Ј and 3Ј ends of each of the strands (14). cccDNA is the template for transcription of...

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Section: Discussionmentioning

confidence: 99%

Roles of the Envelope Proteins in the Amplification of Covalently Closed Circular DNA and Completion of Synthesis of the Plus-Strand DNA in Hepatitis B Virus

Lentz

Loeb

2011

J Virol

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“…In addition, despite the recent discovery of the putative receptor of HBV, we still lack a robust HBV in vitro infection system. In vitro HBV infection efficiency is often poor and the level of HBV replication is low (Gripon et al 2002;Hantz et al 2009). Equally, animal models of hepadnavirus infections are plagued by ethical issues and high costs (chimpanzees), the scarcity of reagents to analyze immunological events (woodchucks, ducks) (Roggendorf and Tolle 1995;Guy et al 2008), or technical difficulties involved with the production of human livers in chimeric mice (Dandri et al 2001;Jo et al 2013).…”

Section: Innate Immunity During Hbv Infection: Recognition Defect or mentioning

confidence: 99%

Immune Response in Hepatitis B Virus Infection

Tan

Koh

Bertoletti

2015

Cold Spring Harb Perspect Med

105

104

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Hepatitis B virus (HBV) can replicate within hepatocytes without causing direct cell damage. The host immune response is, therefore, not only essential to control the spread of virus infection, but it is also responsible for the inflammatory events causing liver pathologies. In this review, we discuss how HBV deals with host immunity and how we can harness it to achieve virus control and suppress liver damage.

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“…HepaRG is a hepatoma cell line that is susceptible to HBV infection (10), but the susceptibility of HepaRG cells to HBV is strictly dependent on the differentiation state induced by DMSO, causing variable toxic side effects and irreproducible results for HBV replication levels. Moreover, poor viral replication, low viral yields, the absence of reinfection, and lack of cccDNA amplification in HepaRG cells make the study of HBV lifecycle and pathogenesis difficult (10). For reasons that still are unknown, HBV clinical isolates do not propagate in cell culture.…”

mentioning

confidence: 99%

“…Several human hepatoma cell lines support HBV replication after HBV DNA transfection, and overexpression of sodium-taurocholate cotransporting polypeptide (NTCP) in HepG2 and Huh7 cells can render these cells able to support HBV produced in cell culture at low efficiency (5,8,9). HepaRG is a hepatoma cell line that is susceptible to HBV infection (10), but the susceptibility of HepaRG cells to HBV is strictly dependent on the differentiation state induced by DMSO, causing variable toxic side effects and irreproducible results for HBV replication levels. Moreover, poor viral replication, low viral yields, the absence of reinfection, and lack of cccDNA amplification in HepaRG cells make the study of HBV lifecycle and pathogenesis difficult (10).…”

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confidence: 99%

Complete replication of hepatitis B virus and hepatitis C virus in a newly developed hepatoma cell line

Yang

Zuo

Wang

et al. 2014

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

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The absence of a robust cell culture system for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection has limited the analysis of the virus lifecycle and drug discovery. We have established a hepatoma cell line, HLCZ01, the first cell line, to the authors' knowledge, supporting the entire lifecycle of both HBV and HCV. HBV surface antigen (HBsAg)-positive particles can be observed in the supernatant and the lumen of the endoplasmic reticulum of the cells via electron microscopy. Interestingly, HBV and HCV clinical isolates propagate in HLCZ01 cells. Both viruses replicate in the cells without evidence of overt interference. HBV and HCV entry are blocked by antibodies against HBsAg and human CD81, respectively, and the replication of HBV and HCV is inhibited by antivirals. HLCZ01 cells mount an innate immune response to virus infection. The cell line provides a powerful tool for exploring the mechanisms of virus entry and replication and the interaction between host and virus, facilitating the development of novel antiviral agents and vaccines.cell culture model | primary human hepatocytes | cccDNA | interferon | ISGs M ore than 500 million people worldwide are persistently infected with hepatitis B virus (HBV) and/or hepatitis B virus (HCV) and are at risk of developing chronic liver diseases (1). There is no vaccine against HCV, and many patients who are persistently infected by HBV or HCV do not respond to currently available therapies (2, 3). Improved understanding of the biology and pathogenesis of these infections is required for the development of vaccine and antiviral drugs (4). The inability to grow HBV and HCV efficiently in cell culture has presented a major obstacle to understanding the virus lifecycle and pathogenesis and to developing improved therapeutics.HBV is a member of the hepadnavirus families, and its genome is a relaxed circular, partially double-stranded DNA molecule. The negative strand has an invariable length of ∼3.2 kb, and the positive strand is 50-100% of this length. Several key issues about the biology of HBV remain to be explored, including the identification of the cellular receptors, the role of the X gene, and the mechanisms by which the viral minichromosome is formed. Covalently closed circular DNA (cccDNA) is responsible for the establishment of viral infection and persistence. Understanding the mechanisms underlying cccDNA formation and regulation is critical for understanding the HBV pathogenesis and finding a cure for hepatitis B. HepG2.2.15 cells derived from the hepatoma cell line HepG2 transfected with the full genome of HBV have been used to study HBV replication (5). Primary human hepatocytes (PHH) are susceptible to HBV infection (6, 7), but the use of this model is hampered by the limited availability and unpredictable variability of human liver. Several human hepatoma cell lines support HBV replication after HBV DNA transfection, and overexpression of sodium-taurocholate cotransporting polypeptide (NTCP) in HepG2 and Huh7 cells can render these cells able t...

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Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells

Cited by 129 publications

References 42 publications

Roles of the Envelope Proteins in the Amplification of Covalently Closed Circular DNA and Completion of Synthesis of the Plus-Strand DNA in Hepatitis B Virus

Roles of the Envelope Proteins in the Amplification of Covalently Closed Circular DNA and Completion of Synthesis of the Plus-Strand DNA in Hepatitis B Virus

Immune Response in Hepatitis B Virus Infection

Complete replication of hepatitis B virus and hepatitis C virus in a newly developed hepatoma cell line

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