scholarly journals Intrahepatic Induction of Alpha/Beta Interferon Eliminates Viral RNA-Containing Capsids in Hepatitis B Virus Transgenic Mice

2000 ◽  
Vol 74 (9) ◽  
pp. 4165-4173 ◽  
Author(s):  
Stefan F. Wieland ◽  
Luca G. Guidotti ◽  
Francis V. Chisari
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Transgenic Mice ◽  
Hbv Dna ◽  
Hbv Replication ◽  
Beta Interferon ◽  
Polycytidylic Acid ◽  
B Virus ◽  
Replicative Intermediates ◽  
Alpha Beta

ABSTRACT We have previously shown that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by stimuli that induce alpha/beta interferon (IFN-α/β) in the liver. The present study was done to identify the step(s) in HBV replication that is affected by this cytokine in transgenic mice treated with the IFN-α/β inducer polyinosinic-polycytidylic acid [poly(I-C)]. Here we show that the pool of cytoplasmic HBV pregenomic RNA (pgRNA)-containing capsids is reduced 10-fold within 9 h after poly(I-C) administration, while there is no change in the abundance of HBV mRNA or in the translational status of cytoplasmic HBV transcripts. In addition, we show that the pool of HBV DNA-containing capsids is not reduced to the same degree until at least 15 h posttreatment, and we show that virus export is not accelerated and the half-life of virions in the serum is unchanged. These results indicate that IFN-α/β triggers intracellular events that either inhibit the assembly of pgRNA-containing capsids or accelerate their degradation, and that maturation and secretion of virus is responsible for clearance of HBV capsids and their cargo of replicative intermediates from the cytoplasm of the hepatocyte.

scholarly journals Nitric Oxide Inhibits Hepatitis B Virus Replication in the Livers of Transgenic Mice

2000 ◽  
Vol 191 (7) ◽  
pp. 1247-1252 ◽  
Author(s):  
Luca G. Guidotti ◽  
Heike McClary ◽  
Jacquelyn Moorhead Loudis ◽  
Francis V. Chisari
Keyword(s):  
Nitric Oxide ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Antiviral Activity ◽  
Transgenic Mice ◽  
Hbv Replication ◽  
Polycytidylic Acid ◽  
B Virus ◽  
Ifn Γ ◽  
Deficient Mice

We have previously identified two antiviral cytokines (interferon [IFN]-γ and IFN-α/β) that downregulate hepatitis B virus (HBV) replication in the liver of transgenic mice. The cytokine-inducible downstream events that inhibit HBV replication have not been identified. One possible factor is nitric oxide (NO), a pleiotropic free radical with antiviral activity that is produced in the liver by the inducible NO synthase (iNOS). To examine the role of NO in our model, we crossed transgenic mice that replicate HBV with mice that lack a functional iNOS. Importantly, iNOS-deficient mice were almost completely resistant to the noncytopathic inhibitory effect of HBV-specific cytotoxic T lymphocytes on viral replication, an effect that we have shown previously to depend on the intrahepatic induction of IFN-γ. Conversely, iNOS-deficient mice were not resistant to the antiviral effect of IFN-α/β induced by either polyinosinic-polycytidylic acid complex or by lymphocytic choriomeningitis virus (LCMV) infection. These results indicate that NO mediates the antiviral activity of IFN-γ, whereas the antiviral activity of IFN-α/β is NO independent. We also compared the relative sensitivity of LCMV to control by NO in these animals. Interestingly, LCMV replicated to higher levels in the liver of iNOS-deficient mice than control mice, indicating that NO controls LCMV replication in the liver, as well as HBV.

scholarly journals Use of the Hepatitis B Virus Recombinant Baculovirus-HepG2 System to Study the Effects of (−)-β-2′,3′-Dideoxy-3′-Thiacytidine on Replication of Hepatitis B Virus and Accumulation of Covalently Closed Circular DNA

1999 ◽  
Vol 43 (8) ◽  
pp. 2017-2026 ◽  
Author(s):  
William E. Delaney ◽  
Thomas G. Miller ◽  
Harriet C. Isom
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hepg2 Cells ◽  
Hbv Dna ◽  
Model Systems ◽  
Extracellular Dna ◽  
Hbv Replication ◽  
B Virus ◽  
Replicative Intermediates

ABSTRACT (−)-β-2′,3′-Dideoxy-3′-thiacytidine (lamivudine [3TC]) is a nucleoside analog which effectively interferes with the replication of hepatitis B virus (HBV) DNA in vitro and in vivo. We have investigated the antiviral properties of 3TC in vitro in HepG2 cells infected with recombinant HBV baculovirus. Different types of information can be obtained with the HBV baculovirus-HepG2 system because (i) experiments can be carried out at various levels of HBV replication including levels significantly higher than those that can be obtained from conventional HBV-expressing cell lines, (ii) cultures can be manipulated and/or treated prior to or during the initiation of HBV expression, and (iii) high levels of HBV replication allow the rapid detection of HBV products including covalently closed circular (CCC) HBV DNA from low numbers of HepG2 cells. The treatment of HBV baculovirus-infected HepG2 cells with 3TC resulted in an inhibition of HBV replication, evidenced by reductions in the levels of both extracellular HBV DNA and intracellular replicative intermediates. The effect of 3TC on HBV replication was both dose and time dependent, and the reductions in extracellular HBV DNA that we observed agreed well with the previously reported efficacy of 3TC in vitro. As expected, levels of HBV transcripts and extracellular hepatitis B surface antigen and e antigen were not affected by 3TC. Importantly, the HBV baculovirus-HepG2 system made it possible to observe for the first time that CCC HBV DNA levels are lower in cells treated with 3TC than in control cells. We also observed that the treatment of HepG2 cells prior to HBV baculovirus infection resulted in a slight increase in the efficacy of 3TC compared to treatments starting 24 h postinfection. The treatment of HepG2 cells with the highest concentration of 3TC tested in this study (2 μM) prior to the initiation of HBV replication markedly inhibited the accumulation of CCC DNA, whereas treatment with the same concentration of 3TC at a time when CCC HBV DNA pools were established within the cells was considerably less effective. In addition, our results suggest that in HepG2 cells, non-protein-associated relaxed circular HBV DNA and particularly CCC HBV DNA are considerably more resistant to 3TC treatment than other forms of HBV DNA, including replicative intermediates and extracellular DNA. We conclude from these studies that the HBV baculovirus-HepG2 system has specific advantages for drug studies and can be used to complement other in vitro model systems currently used for testing antiviral compounds.

scholarly journals Phosphorothioate Di- and Trinucleotides as a Novel Class of Anti-Hepatitis B Virus Agents

2004 ◽  
Vol 48 (6) ◽  
pp. 2199-2205 ◽  
Author(s):  
Radhakrishnan P. Iyer ◽  
Yi Jin ◽  
Arlene Roland ◽  
John D. Morrey ◽  
Samir Mounir ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nucleoside Analogs ◽  
Hbv Dna ◽  
Parallel Synthesis ◽  
Hbv Replication ◽  
Long Term Treatment ◽  
B Virus ◽  
Dna Strand

ABSTRACT Several nucleoside analogs are under clinical development for use against hepatitis B virus (HBV). Lamivudine (3TC), a nucleoside analog, and adefovir dipivoxil (ADV), an acyclonucleotide analog, are clinically approved. However, long-term treatment can induce viral resistance, and following the cessation of therapy, viral rebound is frequently observed. There continues to be a need for new antiviral agents with novel mechanisms of action. A library of more than 600 di- and trinucleotide compounds synthesized by parallel synthesis using a combinatorial strategy was screened for potential inhibitors of HBV replication using the chronically HBV-producing cell line 2.2.15. Through an iterative process of synthesis, lead optimization, and screening, three analogs were identified as potent inhibitors of HBV replication: dinucleotides ORI-7246 (drug concentration at which a 10-fold reduction of HBV DNA was observed [EC90], 1.4 μM) and ORI-9020 (EC90, 1.2 μM) and trinucleotide ORI-7170 (EC90, 7.2 μM). These analogs inhibited the replication of both strands of HBV DNA. No suppression of HBV protein synthesis or intracellular core particle formation by these analogs was observed. No inhibition of HBV DNA strand elongation by the analogs or their 5′-triphosphate versions was apparent in in vitro polymerase assays. Although the exact mechanism of action is not yet identified, present data are consistent with an inhibition of the HBV reverse transcriptase-directed priming step prior to elongation of the first viral DNA strand. In transient-transfection assays, these analogs inhibited the replication of 3TC-resistant HBV. Synergistic interactions in combination treatments between the analogs and either 3TC or ADV were observed. These compounds represent a novel class of anti-HBV molecules and warrant further investigation as potential therapeutic agents.

scholarly journals Inhibition of Cellular Proteasome Activities Mediates HBX-Independent Hepatitis B Virus Replication In Vivo

2010 ◽  
Vol 84 (18) ◽  
pp. 9326-9331 ◽  
Author(s):  
Zhensheng Zhang ◽  
Eun Sun ◽  
Jing-hsiung James Ou ◽  
T. Jake Liang
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Transgenic Mice ◽  
Productive Infection ◽  
Minor Effect ◽  
Hbv Replication ◽  
B Virus ◽  
Culture Models ◽  
A Minor

ABSTRACT The X protein (HBX) of the hepatitis B virus (HBV) is essential for HBV productive infection in vivo. Our previous study (Z. Hu, Z. Zhang, E. Doo, O. Coux, A. L. Goldberg, and T. J. Liang, J. Virol. 73:7231-7240, 1999) shows that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. Previously, we demonstrated that HBX affects hepadnaviral replication through a proteasome-dependent pathway in cell culture models. In the present study, we studied the effect of the proteasome inhibitor MLN-273 in two HBV mouse models. We demonstrated that administration of MLN-273 to transgenic mice containing the replication-competent HBV genome with the defective HBX gene substantially enhanced HBV replication, while the compound had a minor effect on wild-type HBV transgenic mice. Similar results were obtained by using C57BL/6 mice infected with recombinant adenoviruses expressing the replicating HBV genome. Our data suggest that HBV replication is subjected to regulation by cellular proteasome and HBX functions through the inhibition of proteasome activities to enhance HBV replication in vivo.

scholarly journals Hepatocyte Endoplasmic Reticulum Stress Inhibits Hepatitis B Virus Secretion and Delays Intracellular Hepatitis B Virus Clearance After Entecavir Treatment

2021 ◽  
Vol 7 ◽  
Author(s):  
Huan Chen ◽  
Maoyuan Mu ◽  
Qichuan Liu ◽  
Han Hu ◽  
Caiyun Tian ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Er Stress ◽  
Western Blotting ◽  
Hbv Dna ◽  
Translation Initiation Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hbv Replication ◽  
B Virus

Background: The aim of this study was to explore the effects of endoplasmic reticulum (ER) stress on hepatitis B virus (HBV) replication and the antiviral effect of entecavir (ETV).Methods: Thapsigargin (TG) and stearic acid (SA) were used to induce ER stress in HepG2.2.15 cells and HepAD38 cells that contained an integrated HBV genome, while ETV was used to inhibit HBV replication. The expression levels of glucose-regulated protein 78 (GRP78) and phosphorylated eukaryotic translation initiation factor 2 subunit alpha (p-eIF2α) were measured by western blotting. Intracellular HBV DNA was determined by qPCR; HBsAg by western blotting; HBV RNA by real-time RT-qPCR; HBsAg and HBeAg in supernatants by enzyme-linked immunosorbent assay (ELISA); and HBV DNA in supernatants by qPCR.Results: TG and SA induced ER stress in HepG2.2.15 cells and HepAD38 cells from 12 to 48 h post treatment. However, 4-phenylbutyric acid (PBA) partly alleviated the TG-induced ER stress. Moreover, TG inhibited HBsAg, HBeAg, and HBV DNA secretion from 12 to 48 h, while different concentrations of SA inhibited HBsAg and HBV DNA secretion at 48 h. TG promoted intracellular HBV DNA and HBsAg accumulation and the transcription of the HBV 3.5-kb mRNA and S mRNA. PBA treatment restored the secretion of HBsAg and HBV DNA. Finally, ER stress accelerated extracellular HBV DNA clearance but delayed intracellular HBV DNA clearance after ETV treatment.Conclusions: Hepatocyte ER stress promoted intracellular HBV DNA and HBsAg accumulation by inhibiting their secretion. Our study also suggested that hepatocyte ER stress delayed intracellular HBV DNA clearance after ETV treatment.

scholarly journals Rebound of Hepatitis B Virus Replication in HepG2 Cells after Cessation of Antiviral Treatment

2002 ◽  
Vol 76 (16) ◽  
pp. 8148-8160 ◽  
Author(s):  
Ayman M. Abdelhamed ◽  
Colleen M. Kelley ◽  
Thomas G. Miller ◽  
Phillip A. Furman ◽  
Harriet C. Isom
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Drug Treatment ◽  
Time Course ◽  
Antiviral Treatment ◽  
Recombinant Baculovirus ◽  
Antiviral Agent ◽  
Hbv Dna ◽  
Hbv Replication ◽  
B Virus

ABSTRACT Treatment of patients with lamivudine (3TC) results in loss of detectable levels of hepatitis B virus (HBV) DNA from serum; however, the relapse rate, with regard to both reappearance of virus in the bloodstream and hepatic inflammation, is high when therapy is terminated. Although the rebound observed in patients has also been seen in animal hepadnavirus models, rebound has not been analyzed in an in vitro cell culture system. In this study, we used the HBV recombinant baculovirus/HepG2 system to measure the time course of antiviral agent-mediated loss of HBV replication as well as the time course and magnitude of HBV production after release from antiviral treatment. Because of the sensitivity of the system, it was possible to measure secreted virions, intracellular replicative intermediates, and nuclear non-protein-bound HBV DNA and separately analyze individual species of DNA, such as single-stranded HBV DNA compared to the double-stranded form and relaxed circular compared to covalently closed circular HBV DNA. We first determined that HBV replication in the HBV recombinant baculovirus/HepG2 system could proceed for at least 35 days, with a 30-day plateau level of replication, making it possible to study antiviral agent-mediated loss of HBV followed by rebound after cessation of drug treatment. All HBV DNA species decreased in a time-dependent fashion following antiviral treatment, but the magnitude of decline differed for each HBV DNA species, with the covalently closed circular form of HBV DNA being the most resistant to drug therapy. When drug treatment ceased, HBV DNA species reappeared with a pattern that recapitulated the initiation of replication, but with a different time course.

scholarly journals Strong and Selective Inhibitors of Hepatitis B Virus Replication among Novel N4-Hydroxy- and 5-Methyl-β-l-Deoxycytidine Analogues

2007 ◽  
Vol 51 (7) ◽  
pp. 2523-2530 ◽  
Author(s):  
E. Matthes ◽  
A. Funk ◽  
I. Krahn ◽  
K. Gaertner ◽  
M. von Janta-Lipinski ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Inhibitory Concentration ◽  
Hbv Dna ◽  
Selective Inhibitor ◽  
Effective Concentration ◽  
Active Inhibitor ◽  
Hbv Replication ◽  
B Virus

ABSTRACT Novel N4-hydroxy- and 5-methyl-modified β-l-deoxycytidine analogues were synthesized and evaluated as anti-hepatitis B virus (HBV) agents. Their in vitro efficiencies were investigated in HepG2.2.15 cells stably transfected with HBV. β-l-2′,3′-Didehydro-2′,3′-dideoxy-N4-hydroxycytidine (β-l-Hyd4C) was most effective in reducing secreted HBV DNA (50% effective concentration [EC50], 0.03 μM), followed by β-l-2′,3′-dideoxy-3′-thia-N4-hydroxycytidine (EC50, 0.51 μM), β-l-2′,3′-dideoxy-N4-hydroxycytidine (EC50, 0.55 μM), and β-l-5-methyl-2′-deoxycytidine (EC50, 0.9 μM). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the β-l-cytidine derivatives was also assessed. In accordance with the cell culture data, β-l-Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 μM. The cytotoxicities of some of the 4-NHOH-modified β-l-nucleosides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH2 group. The 50% cytotoxic concentrations for β-l-Hyd4C in HepG2 and HL-60 cells were 2,500 μM and 3,500 μM, respectively. In summary, our results demonstrate that at least β-l-Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.

scholarly journals Enhancement of Hepatitis B Virus Replication by Its X Protein in Transgenic Mice

2002 ◽  
Vol 76 (5) ◽  
pp. 2579-2584 ◽  
Author(s):  
Zhenming Xu ◽  
T. S. Benedict Yen ◽  
Lanying Wu ◽  
Charles R. Madden ◽  
Wenjie Tan ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Transgenic Mice ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
X Protein ◽  
Multifunctional Protein ◽  
Hbv Replication ◽  
B Virus

ABSTRACT Hepatitis B virus (HBV) X gene encodes a multifunctional protein that can regulate cellular signaling pathways, interact with cellular transcription factors, and induce hepatocellular oncogenesis. In spite of its diverse activities, the precise role of the X protein in the viral life cycle of HBV remains unclear. To investigate this question, we have produced transgenic mice that carry either the wild-type HBV genome or a mutated HBV genome incapable of expressing the 16.5-kDa X protein. Our results indicate that while the X protein is not absolutely essential for HBV replication or its maturation in transgenic mice, it can enhance viral replication, apparently by activating viral gene expression. These results demonstrate a transactivation role of the X protein in HBV replication in transgenic mice.

scholarly journals Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus.

1997 ◽  
Vol 41 (7) ◽  
pp. 1444-1448 ◽  
Author(s):  
S F Innaimo ◽  
M Seifer ◽  
G S Bisacchi ◽  
D N Standring ◽  
R Zahler ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Line ◽  
Nucleoside Analogs ◽  
Hbv Replication ◽  
Mitochondrial Dna Content ◽  
B Virus ◽  
Antiviral Activities ◽  
Replicative Intermediates

BMS-200475 is a novel carbocyclic 2'-deoxyguanosine analog found to possess potent and selective anti-hepatitis B virus (anti-HBV) activity. BMS-200475 is distinguished from guanosine by replacement of the natural furanose oxygen on the sugar moiety with an exo carbon-carbon double bond. In the HepG2 stably transfected cell line 2.2.15, BMS-200475 had a 50% effective concentration (EC50) of 3.75 nM against HBV, as determined by analysis of secreted HBV DNA. Structurally related compounds with adenine, iodouracil, or thymine base substitutions were significantly less potent or were inactive. Direct comparison of the antiviral activities of BMS-200475 with those of a variety of other nucleoside analogs, including lamivudine (EC50 = 116.26 nM), demonstrated the clearly superior in vitro potency of BMS-200475 in 2.2.15 cells. Intracellular HBV replicative intermediates were uniformly reduced when cells were treated with BMS-200475, but rebounded after treatment was terminated. The concentration of BMS-200475 causing 50% cytotoxicity in 2.2.15 cell cultures was 30 microM, approximately 8,000-fold greater than the concentration required to inhibit HBV replication in the same cell line. Treatment with BMS-200475 resulted in no apparent inhibitory effects on mitochondrial DNA content.

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