Effect of interferon alpha on hepatitis B virus replication and gene expression in transiently transfected human hepatoma cells

Rang, Andreas; Günther, Stephan; Will, Hans

doi:10.1016/s0168-8278(99)80279-7

Cited by 60 publications

(54 citation statements)

References 34 publications

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“…Although similar findings have been obtained by others (17), and IFN-␣/␤ has been reported to inhibit HBV gene expression in other systems in vitro (7,26,27,32), all previous studies have been performed with poorly differentiated, transformed hepatoma cell lines that may not reflect the intracellular events triggered by cytokines in well-differentiated hepatocytes.…”

Section: Discussionsupporting

confidence: 80%

Cytokine-Sensitive Replication of Hepatitis B Virus in Immortalized Mouse Hepatocyte Cultures

Pasquetto

Wieland

Uprichard

et al. 2002

J Virol

119

120

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We have previously shown that alpha/beta interferon (IFN-␣/␤) and gamma interferon (IFN-␥) inhibit hepatitis B virus (HBV) replication by eliminating pregenomic RNA containing viral capsids from the hepatocyte. We have also shown that HBV-specific cytotoxic T lymphocytes that induce IFN-␥ and tumor necrosis factor alpha (TNF-␣) in the liver can inhibit HBV gene expression by destabilizing preformed viral mRNA. In order to further study the antiviral activity of IFN-␣/␤, IFN-␥, and TNF-␣ at the molecular level, we sought to reproduce these observations in an in vitro system. Accordingly, hepatocytes were derived from the livers of HBV-transgenic mice that also expressed the constitutively active cytoplasmic domain of the human hepatocyte growth factor receptor (c-Met). Here, we show that the resultant well-differentiated, continuous hepatocyte cell lines (HBV-Met) replicate HBV and that viral replication in these cells is efficiently controlled by IFN-␣/␤ or IFN-␥, which eliminate pregenomic RNA-containing capsids from the cells as they do in the liver. Furthermore, we demonstrate that IFN-␥, but not IFN-␣/␤, is capable of inhibiting HBV gene expression in this system, especially when it acts synergistically with TNF-␣. These cells should facilitate the analysis of the intracellular signaling pathways and effector mechanisms responsible for these antiviral effects.Hepatitis B virus (HBV) is a hepatotropic DNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma (5). We have previously shown that the intrahepatic induction of alpha/beta interferon (IFN-␣/␤), gamma interferon (IFN-␥), and tumor necrosis factor alpha (TNF-␣) by various stimuli can inhibit HBV gene expression and/or HBV replication in the livers of HBV-transgenic mice (3,9,10,23). More recently, we demonstrated that similar noncytopathic antiviral events probably occur in the livers of chimpanzees acutely infected with HBV (15).Although it is now well documented that HBV replication can be inhibited by these cytokines (11,19), in vivo systems are by nature difficult to manipulate and control. Therefore, studies designed to determine which cytokines primarily inhibit HBV replication and to decipher the antiviral intracellular mechanism(s) they induce would be greatly facilitated by an in vitro cell culture system that accurately reflects the cytokinedependent control of HBV observed in vivo. Unfortunately, primary cultures of HBV-transgenic hepatocytes are not suitable for these studies because they rapidly become less well differentiated in vitro. Recently, however, well-differentiated immortalized mouse hepatocyte cell lines were established from transgenic mice expressing the constitutively active cytoplasmic domain of the human hepatocyte growth factor receptor (c-Met) in their livers (2). Cells prepared from these mice are not transformed, and they can be induced to a stable, highly differentiated hepatocyte phenotype by dimethyl sulfoxide (DMSO) (29).In this report, we describe the establishment and characterization of an i...

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Section: Discussionsupporting

confidence: 80%

Cytokine-Sensitive Replication of Hepatitis B Virus in Immortalized Mouse Hepatocyte Cultures

Pasquetto

Wieland

Uprichard

et al. 2002

J Virol

119

120

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“…34 The addition of recombinant interferon alfa to lamivudine therapy allowed us to obtain a significant decrease in viral DNA titers in all 3 patients. As interferon alfa has multiple sites of action in the viral life cycle independently of the reverse transcriptase activity, 46,47 these preliminary results may indicate a potential antiviral effect of interferon alfa on YMDD mutant replication. Because add-on therapy with interferon alfa was more intensive, using daily administration or a combination with famciclovir, than in the previous course of interferon alfa, it is difficult to speculate that the lamivudine-resistant strains are more sensitive to interferon alfa than Comparison with historic biopsy was done, a variation of 1 or more point of Knodell score was considered as worsening (*) or improvement ( †).…”

Section: Discussionmentioning

confidence: 81%

Early Detection of Viral Resistance by Determination of Hepatitis B Virus Polymerase Mutations in Patients Treated by Lamivudine for Chronic Hepatitis B

et al. 2000

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We have analyzed the molecular dynamics of emergence of drug-resistant strains in patients receiving lamivudine therapy for chronic hepatitis B. Twenty consecutive patients with lamivudine resistance were studied ( Major advances in the antiviral treatment of chronic hepatitis B have been made with the development of new antiviral compounds that inhibit the reverse transcriptase activity of hepatitis B virus (HBV). Recently, lamivudine has been approved for the therapy of chronic hepatitis B. Results of clinical trials in patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B showed that lamivudine administration was associated with an increased rate of HBV-DNA clearance and antibody to hepatitis B e antigen (anti-HBe) seroconversion together with a significant improvement in liver histology, by comparison with patients who received placebo. [1][2][3][4] In patients infected with a precore mutant associated with an HBeAg-negative chronic hepatitis B, lamivudine therapy for 12 months was also associated with HBV-DNA suppression, normalization of alanine transaminase (ALT) levels, and improvement in liver histology in approximately 60% of patients. 5 In liver transplant recipients with HBV recurrence on the liver graft, lamivudine therapy for 52 weeks allowed suppression of viral replication in 60% of patients. 6 However, in immune competent patients, selection of lamivudine-resistant strains, which may lead to failure of antiviral therapy of chronic hepatitis B, occurs in approximately 16% to 43% of the patients treated for 1 year. 2-5 Selection of drugresistant mutants has also been observed in human immunodeficiency virus (HIV)-HBV coinfected patients treated with lamivudine for their HIV infection 7 and in liver transplant recipients treated with lamivudine for HBV recurrence on the liver graft. 6 Drug resistance is associated with a rise in serum HBV-DNA titers above the detection limit of the assay, which is commonly referred to as phenotypic resistance. In these trials, appearance of viral resistance starts after 6 months of treatment and increases with the duration of treatment. Despite phenotypic resistance, HBV-DNA levels and ALT levels remained lower by comparison with baseline values. In the majority of these patients, lamivudine resistance was not associated with significant clinical or histologic worsening. [2][3][4]6 However, some patients showed clinical deterioration with liver failure, especially in the liver transplantation setting. 6,8 Viral resistance is associated with the selection of major mutations in the YMDD motif within the C domain of the viral reverse transcriptase, and in the conserved B domain. These mutants have been classified in group I with B and C domain Abbreviations: HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; anti-HBe, antibody to hepatitis B e antigen; ALT, alanine transaminase; HIV, human immunodeficiency virus; ULN, upper limit of normal; bDNA, branched DNA; PCR, polymerase chain reaction; HBsAg, hepatitis B surface antigen.From 1 INSERM Unit 2...

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“…HBV expression plasmid (1.5 g) was co-transfected with 0.5 g of pCMV/SEAP (kindly provided by J. Taylor, Philadelphia), which codes for a secreted alkaline phosphatase, or, if indicated, with 0.07 g of pEGFP-N1. To monitor transfection efficiency, Northern blot membranes were hybridized with a GFP mRNA-specific probe, or the amount of secreted alkaline phosphatase activity secreted into the medium of transfected cells was determined as described previously (9).…”

Section: Methodsmentioning

confidence: 99%

“…A number of publications highlighted the potential of cytokines to induce the post-transcriptional downregulation of HBV RNA (for review see Ref. 7) in an HBV transgenic mouse model (8), in human hepatoma cells (9,10), as well as the inhibition of duck HBV replication (11)(12)(13). In the HBV transgenic mouse model, the cytotoxic T lymphocyte (CTL) response to HBV antigens was shown to inhibit HBV replication by a post-transcriptional, noncytotoxic mechanism, leading to effective viral RNA degradation as induced by interferon-␥ and tumor necrosis factor-␣ (8,14,15).…”

mentioning

confidence: 99%

Functional Characterization of the Interaction between Human La and Hepatitis B Virus RNA

Ehlers

Horke

Reumann

et al. 2004

Journal of Biological Chemistry

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The La protein is a multifunctional RNA-binding protein and has also been suggested to be involved in the stabilization of hepatitis B virus (HBV) RNA. Here we demonstrate that antibodies against the human La protein specifically precipitate HBV RNA from HBV ribonucleoprotein-containing mammalian cell extracts, providing evidence for the association between human La and HBV RNA. Moreover, we report that the turnover of HBV RNA depends on structural features and less on the primary sequence of the La-binding site on the viral RNA. In addition we show that the interaction between human La and HBV RNA in vitro is modulated by accessory factor(s) in a phosphorylation-dependent manner. Taken together these data indicate that both structural features, the composition of La/HBV ribonucleoprotein particles as well as interacting cellular factors, are critical determinants in the regulation of the stability of the HBV RNA.RNA metabolism depends on the formation of ribonucleoprotein particles mediating diverse processes such as splicing, polyadenylation, nuclear export, and the regulation of mRNA stability (1, 2). The formation of RNPs 1 is a tightly controlled process, potentially regulated by several stimuli, including hormones and cytokines. Such stimuli can alter the RNA binding activity of proteins on the post-translational level by phosphorylation or dephosphorylation and thereby the processing and stability of RNAs (3-5). In addition, RNA processing depends on various cis-acting elements including splice sites, export elements, and endoribonucleolytic cleavage sites recognized by RNA-binding proteins. To understand fully the regulation of processing of a specific RNA, both trans-acting factors and cis-acting elements as well as their functions need to be known. The same applies for a detailed understanding of the metabolism of viral RNA. Such studies could lead to the identification of novel cellular targets valuable for the development of innovative antiviral strategies when focused on the post-transcriptional control of RNAs of viruses with global medical importance. This applies to hepatitis B virus (HBV) with more than 300 million chronically infected carriers worldwide who await more effective antiviral therapies.HBV is a noncytopathic, hepatotropic virus with a 3.2-kb circular DNA genome. After conversion into a covalently closed circular DNA, this genome serves in the nucleus as a template for transcription of all viral RNAs. Synthesis of these transcripts is driven by at least four promotors, leading to a large size heterogeneity with many different 5Ј-ends, whereas they all have very similar 3Ј-ends because of processing at the same polyadenylylation site (6). The so-called pregenomic RNA (slightly longer than genome length) is encapsidated into nucleocapsids where it is reverse-transcribed into viral DNA. This RNA serves also as messenger for synthesis of the viral P protein as well as for the core protein. The viral surface proteins as well as a regulatory protein with a role in hepatocarcinogenesis, des...

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Effect of interferon alpha on hepatitis B virus replication and gene expression in transiently transfected human hepatoma cells

Cited by 60 publications

References 34 publications

Cytokine-Sensitive Replication of Hepatitis B Virus in Immortalized Mouse Hepatocyte Cultures

Cytokine-Sensitive Replication of Hepatitis B Virus in Immortalized Mouse Hepatocyte Cultures

Early Detection of Viral Resistance by Determination of Hepatitis B Virus Polymerase Mutations in Patients Treated by Lamivudine for Chronic Hepatitis B

Functional Characterization of the Interaction between Human La and Hepatitis B Virus RNA

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