Inhibition of Hepatitis B Virus (HBV) Replication by Pyrimidines Bearing an Acyclic Moiety:  Effect on Wild-Type and Mutant HBV

2006 ◽  
Vol 49 (6) ◽  
pp. 2049-2054 ◽  
Author(s):  
Wassila Semaine ◽  
Monika Johar ◽  
D. Lorne J. Tyrrell ◽  
Rakesh Kumar ◽  
B. Agrawal
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Wild Type ◽  
Hbv Replication ◽  
B Virus

scholarly journals Efficacies of Entecavir against Lamivudine-Resistant Hepatitis B Virus Replication and Recombinant Polymerases In Vitro

2002 ◽  
Vol 46 (8) ◽  
pp. 2525-2532 ◽  
Author(s):  
S. Levine ◽  
D. Hernandez ◽  
G. Yamanaka ◽  
S. Zhang ◽  
R. Rose ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cross Resistance ◽  
Wild Type ◽  
Resistance Mutations ◽  
Hbv Replication ◽  
B Virus ◽  
Inhibition Studies

ABSTRACT Entecavir (ETV) is a potent and selective inhibitor of hepatitis B virus (HBV) replication in vitro and in vivo that is currently in clinical trials for the treatment of chronic HBV infections. A major limitation of the current HBV antiviral therapy, lamivudine (3TC), is the emergence of drug-resistant HBV in a majority of treated patients due to specific mutations in the nucleotide binding site of HBV DNA polymerase (HBV Pol). To determine the effects of 3TC resistance mutations on inhibition by ETV triphosphate (ETV-TP), a series of in vitro studies were performed. The inhibition of wild-type and 3TC-resistant HBV Pol by ETV-TP was measured using recombinant HBV nucleocapsids, and compared to that of 3TC-TP. These enzyme inhibition studies demonstrated that ETV-TP is a highly potent inhibitor of wild-type HBV Pol and is 100- to 300-fold more potent than 3TC-TP against 3TC-resistant HBV Pol. Cell culture assays were used to gauge the potential for antiviral cross-resistance of 3TC-resistant mutants to ETV. Results demonstrated that ETV inhibited the replication of 3TC-resistant HBV, but 20- to 30-fold higher concentrations were required. To gain further perspective regarding the potential therapeutic use of ETV, its phosphorylation was examined in hepatoma cells treated with extracellular concentrations representative of drug levels in plasma in ETV-treated patients. At these concentrations, intracellular ETV-TP accumulated to levels expected to inhibit the enzyme activity of both wild-type and 3TC-resistant HBV Pol. These findings are predictive of potent antiviral activity of ETV against both wild-type and 3TC-resistant HBV.

scholarly journals Phenylpropenamide Derivatives AT-61 and AT-130 Inhibit Replication of Wild-Type and Lamivudine-Resistant Strains of Hepatitis B Virus In Vitro

2002 ◽  
Vol 46 (9) ◽  
pp. 3057-3060 ◽  
Author(s):  
William E. Delaney ◽  
Ros Edwards ◽  
Danni Colledge ◽  
Tim Shaw ◽  
Phil Furman ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nucleoside Analogue ◽  
Wild Type ◽  
Hbv Replication ◽  
B Virus ◽  
Resistant Strains

ABSTRACT The phenylpropenamide derivatives AT-61 and AT-130 are nonnucleoside analogue inhibitors of hepatitis B virus (HBV) replication. They inhibited the replication of wild-type HBV with 50% inhibitory concentrations of 21.2 ± 9.5 and 2.40 ± 0.92 μM, respectively, compared to 0.064 ± 0.020 μM lamivudine. There were no significant differences in sensitivity between wild-type and nucleoside analogue-resistant (rtL180M, rtM204I, and rtL180M + rtM204V) HBV.

scholarly journals Enhancement of Hepatitis B Virus Replication by the Regulatory X Protein In Vitro and In Vivo

2006 ◽  
Vol 81 (6) ◽  
pp. 2656-2662 ◽  
Author(s):  
Victor V. Keasler ◽  
Amanda J. Hodgson ◽  
Charles R. Madden ◽  
Betty L. Slagle
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Virus Replication ◽  
Hepg2 Cells ◽  
Wild Type ◽  
Tail Vein ◽  
Hbv Replication ◽  
Tail Vein Injection ◽  
B Virus

ABSTRACT The 3.2-kb hepatitis B virus (HBV) genome encodes a single regulatory protein termed HBx. While multiple functions have been identified for HBx in cell culture, its role in virus replication remains undefined. In the present study, we combined an HBV plasmid-based replication assay with the hydrodynamic tail vein injection model to investigate the function(s) of HBx in vivo. Using a greater-than-unit-length HBV plasmid DNA construct (payw1.2) and a similar construct with a stop codon at position 7 of the HBx open reading frame (payw1.2*7), we showed that HBV replication in transfected HepG2 cells was reduced 65% in the absence of HBx. These plasmids were next introduced into the livers of outbred ICR mice via hydrodynamic tail vein injection. At the peak of virus replication, at 4 days postinjection, intrahepatic markers of HBV replication were reduced 72% to 83% in mice injected with HBx-deficient payw1.2*7 compared to those measured in mice receiving wild-type payw1.2. A second plasmid encoding HBx was able to restore virus replication from payw1.2*7 to wild-type levels. Finally, viremia was monitored over the course of acute virus replication, and at 4 days postinjection, it was reduced by nearly 2 logs in the absence of HBx. These studies establish that the role for HBx in virus replication previously shown in transfected HepG2 cells is also apparent in the mouse liver within the context of acute hepatitis. Importantly, the function of HBx can now be studied in an in vivo setting that more closely approximates the cellular environment for HBV replication.

scholarly journals Activation and Inhibition of Cellular Calcium and Tyrosine Kinase Signaling Pathways Identify Targets of the HBx Protein Involved in Hepatitis B Virus Replication

2003 ◽  
Vol 77 (14) ◽  
pp. 7713-7719 ◽  
Author(s):  
Michael J. Bouchard ◽  
Robyn J. Puro ◽  
Lihua Wang ◽  
Robert J. Schneider
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Tyrosine Kinase ◽  
Calcium Signaling ◽  
Signaling Pathways ◽  
Cytosolic Calcium ◽  
Wild Type ◽  
Tyrosine Kinase Signaling ◽  
Hbv Replication ◽  
B Virus

ABSTRACT Human hepatitis B virus (HBV) HBx protein is a multifunctional protein that activates cellular signaling pathways and is thought to be essential for viral infection. Woodchuck HBV mutants that lack HBx are unable to replicate in vivo or are severely impaired. HBV replication in HepG2 cells, a human hepatoblastoma cell line, is stimulated 5- to 10-fold by HBx protein. We have utilized the HepG2, HBx-dependent HBV replication system to study the effects of activators and inhibitors of cytosolic calcium and tyrosine kinase signaling pathways on viral replication. By transfecting either a wild-type HBV genome or an HBV genome that does not express HBx and then treating transfected cells with activators or inhibitors of signaling pathways, we identified compounds that either impair wild-type HBV replication or rescue HBx-deficient HBV replication. Geldanamycin or herbimycin A, tyrosine kinase inhibitors, blocked HBV replication. Derivatives of cyclosporine, i.e., cyclosporine A, cyclosporine H, and SDZ NIM811, which block cytosolic calcium signaling and specifically the mitochondrial permeability transition pore (SDZ NIM811), also impaired HBV replication. Treatment of cells with compounds that increase cytosolic calcium levels by a variety of mechanisms rescued replication of an HBx-deficient HBV mutant. Transcription of viral RNA and production of viral capsids were only minimally affected by these treatments. These results define a functional signaling circuit for HBV replication that includes calcium signaling and activation of cytosolic signaling pathways involving Src kinases, and they suggest that these pathways are stimulated by HBx acting on the mitochondrial transition pore.

scholarly journals Substitution of proline 306 in the reverse transcriptase domain of hepatitis B virus regulates replication

2005 ◽  
Vol 86 (1) ◽  
pp. 85-90 ◽  
Author(s):  
Xu Lin ◽  
Zhang-Mei Ma ◽  
Xin Yao ◽  
Li-Fang He ◽  
Zheng-Hong Yuan ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Wild Type Virus ◽  
Wild Type ◽  
Functional Changes ◽  
Hbv Replication ◽  
B Virus ◽  
Dna Template ◽  
The Impact

The proline residue at position 306 in hepatitis B virus (HBV) reverse transcriptase (rtP306) has been suggested to constrain the conformation of the α-helices in the thumb subdomain that interacts with the viral DNA template-primer. To study the impact of residue rt306 in HBV replication further, 11 site-directed mutants were constructed that substituted rtP306 with different amino acids. The replicative competencies of these mutants were assayed by HepG2 cell transfection and real-time PCR. When rtP306 was substituted with glycine or threonine, the replication competency of these mutants was drastically reduced to 1·96 and 4·51 % of the wild-type HBV level, respectively. When rtP306 was substituted with glutamic acid, the replicative competency of the mutant increased up to 9·4-fold compared with wild-type virus. The results also showed that changes in the replicative competency of these constructed mutants were not associated with functional changes of HBV enhancer I. These results indicate the importance of amino acid(s) at the interface between the thumb and palm subdomains in modulating the replicative competency of HBV isolates. This regulatory residue(s) could serve as a new target for the development of anti-HBV drugs.

Precore/Core Promoter Mutant Hepatitis B Virus Produces More Severe Histologic Liver Disease than Wild Type Hepatitis B Virus

2007 ◽  
Vol 1 (1) ◽  
pp. 41-46 ◽  
Author(s):  
Mamun-Al-Mahtab ◽  
Salimur Rahman ◽  
Mobin Khan ◽  
Ayub Mamun ◽  
Kamal
Keyword(s):  
Liver Disease ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Promoter ◽  
Wild Type ◽  
B Virus ◽  
Promoter Mutant

scholarly journals Hepatitis B Virus Replication Is Associated with an HBx-Dependent Mitochondrion-Regulated Increase in Cytosolic Calcium Levels

2007 ◽  
Vol 81 (21) ◽  
pp. 12061-12065 ◽  
Author(s):  
Stephanie L. McClain ◽  
Amy J. Clippinger ◽  
Rebecca Lizzano ◽  
Michael J. Bouchard
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Molecular Mechanisms ◽  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cytosolic Calcium ◽  
Hbv Replication ◽  
B Virus

ABSTRACT The nonstructural hepatitis B virus (HBV) protein HBx has an important role in HBV replication and in HBV-associated liver disease. Many activities have been linked to HBx expression; however, the molecular mechanisms underlying many of these activities are unknown. One proposed HBx function is the regulation of cytosolic calcium. We analyzed calcium levels in HepG2 cells that expressed HBx or replicating HBV, and we demonstrated that HBx, expressed in the absence of other HBV proteins or in the context of HBV replication, elevates cytosolic calcium. We linked this elevation of cytosolic calcium to the association of HBx with the mitochondrial permeability transition pore.

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.

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