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 Alkoxyalkyl Esters of 9-(S)-(3-Hydroxy-2-Phosphonomethoxypropyl) Adenine Are Potent and Selective Inhibitors of Hepatitis B Virus (HBV) Replication In Vitro and in HBV Transgenic Mice In Vivo

2009 ◽  
Vol 53 (7) ◽  
pp. 2865-2870 ◽  
Author(s):  
John D. Morrey ◽  
Brent E. Korba ◽  
James R. Beadle ◽  
David L. Wyles ◽  
Karl Y. Hostetler
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Antiviral Activity ◽  
Transgenic Mice ◽  
Adefovir Dipivoxil ◽  
Murine Cytomegalovirus ◽  
Cross Resistance ◽  
B Virus

ABSTRACT Alkoxyalkyl esters of acyclic nucleoside phosphonates have previously been shown to have increased antiviral activity when they are administered orally in animal models of viral diseases, including lethal infections with vaccinia virus, cowpox virus, ectromelia virus, murine cytomegalovirus, and adenovirus. 9-(S)-(3-Hydroxy-2-phosphonomethoxypropyl)adenine [(S)-HPMPA] was previously shown to have activity against hepatitis B virus (HBV) in vitro. To assess the effect of alkoxyalkyl esterification of (S)-HPMPA, we prepared the hexadecyloxypropyl (HDP), 15-methyl-hexadecyloxypropyl (15M-HDP), and octadecyloxyethyl (ODE) esters and compared their activities with the activity of adefovir dipivoxil in vitro and in vivo. Alkoxyalkyl esters of (S)-HPMPA were 6 to 20 times more active than unmodified (S)-HPMPA on the basis of their 50% effective concentrations in 2.2.15 cells. The increased antiviral activity appeared to be due in part to the increased uptake and conversion of HDP-(S)-HPMPA to HPMPA diphosphate observed in HepG2 cells in vitro. HDP-(S)-HPMPA retained full activity against HBV mutants resistant to lamivudine (L180M, M204V), but cross-resistance to a mutant resistant to adefovir (N236T) was detected. HDP-(S)-HPMPA is orally bioavailable and provides excellent liver exposure to the drug. Oral treatment of HBV transgenic mice with HDP-(S)-HPMPA, 15M-HDP-(S)-HPMPA, and ODE-(S)-HPMPA for 14 days reduced liver HBV DNA levels by roughly 1.5 log units, a response equivalent to that of adefovir dipivoxil.

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 Hepatitis B Virus pX Targets TFIIB in Transcription Coactivation

1998 ◽  
Vol 18 (3) ◽  
pp. 1562-1569 ◽  
Author(s):  
Izhak Haviv ◽  
Meir Shamay ◽  
Gilad Doitsh ◽  
Yosef Shaul
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Polymerase Ii ◽  
Wild Type ◽  
Nuclear Extracts ◽  
Wild Type Phenotype ◽  
B Virus

ABSTRACT pX, the hepatitis B virus (HBV)-encoded regulator, coactivates transcription through an unknown mechanism. pX interacts with several components of the transcription machinery, including certain activators, TFIIB, TFIIH, and the RNA polymerase II (POLII) enzyme. We show that pX localizes in the nucleus and coimmunoprecipitates with TFIIB from nuclear extracts. We used TFIIB mutants inactive in binding either POLII or TATA binding protein to study the role of TFIIB-pX interaction in transcription coactivation. pX was able to bind the former type of TFIIB mutant and not the latter. Neither of these sets of TFIIB mutants supports transcription. Remarkably, the latter TFIIB mutants fully block pX activity, suggesting the role of TFIIB in pX-mediated coactivation. By contrast, in the presence of pX, TFIIB mutants with disrupted POLII binding acquire the wild-type phenotype, both in vivo and in vitro. These results suggest that pX may establish the otherwise inefficient TFIIB mutant-POLII interaction, by acting as a molecular bridge. Collectively, our results demonstrate that TFIIB is the in vivo target of pX.

scholarly journals In Vitro Susceptibilities of Wild-Type or Drug-Resistant Hepatitis B Virus to (−)-β-d-2,6-Diaminopurine Dioxolane and 2′-Fluoro-5-Methyl-β-l-Arabinofuranosyluracil

2001 ◽  
Vol 45 (9) ◽  
pp. 2495-2501 ◽  
Author(s):  
Ruth Chin ◽  
Tim Shaw ◽  
Joseph Torresi ◽  
Vittina Sozzi ◽  
Christian Trautwein ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Prolonged Treatment ◽  
Cross Resistance ◽  
Drug Resistant ◽  
Wild Type ◽  
Lamivudine Resistance ◽  
Chronic Hepatitis B Virus ◽  
B Virus

ABSTRACT Prolonged treatment of chronic hepatitis B virus (HBV) infection with lamivudine ([−]-β-l-2′,3′-dideoxy-3′ thiacytidine) or famciclovir may select for viral mutants that are drug resistant due to point mutations in the polymerase gene. Determining whether such HBV mutants are sensitive to new antiviral agents is therefore important. We used a transient transfection system to compare the sensitivities of wild-type HBV and four lamivudine- and/or famciclovir-resistant HBV mutants to adefovir [9-(2-phosphonyl-methoxyethyl)-adenine; PMEA] and the nucleoside analogues (−)-β-d-2, 6-diaminopurine dioxolane (DAPD) and 2′-fluoro-5-methyl-β-l-arabinofuranosyluracil (l-FMAU). The drug-resistant mutants contained amino acid substitutions in the polymerase protein. We found that the M550I and M550V plus L526M substitutions, which confer lamivudine resistance, did not confer cross-resistance to adefovir or DAPD, but conferred cross-resistance to l-FMAU. The M550V substitution in isolation conferred a similar phenotype to M550I, except that it did not confer significant resistance to l-FMAU. The L526M substitution, which is associated with famciclovir resistance, conferred cross-resistance to l-FMAU but not to adefovir or DAPD. Inhibition of HBV secretion by DAPD, l-FMAU, and adefovir did not always correlate with inhibition of the generation of intracellular HBV replicative intermediates, suggesting that these analogs may preferentially inhibit specific stages of the viral replication cycle.

scholarly journals Cross-Resistance Testing of Antihepadnaviral Compounds Using Novel Recombinant Baculoviruses Which Encode Drug-Resistant Strains of Hepatitis B Virus

2001 ◽  
Vol 45 (6) ◽  
pp. 1705-1713 ◽  
Author(s):  
William E. Delaney ◽  
Ros Edwards ◽  
Danni Colledge ◽  
Tim Shaw ◽  
Joseph Torresi ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Active Form ◽  
Recombinant Baculoviruses ◽  
Resistance Testing ◽  
Cross Resistance ◽  
Drug Resistant ◽  
Hbv Replication ◽  
B Virus

ABSTRACT Long-term nucleoside analog therapy for hepatitis B virus (HBV)-related disease frequently results in the selection of mutant HBV strains that are resistant to therapy. Molecular studies of such drug-resistant variants are clearly warranted but have been difficult to do because of the lack of convenient and reliable in vitro culture systems for HBV. We previously developed a novel in vitro system for studying HBV replication that relies on the use of recombinant baculoviruses to deliver greater than unit length copies of the HBV genome to HepG2 cells. High levels of HBV replication can be achieved in this system, which has recently been used to assess the effects of lamivudine on HBV replication and covalently closed circular DNA accumulation. The further development of this novel system and its application to determine the cross-resistance profiles of drug-resistant HBV strains are described here. For these studies, novel recombinant HBV baculoviruses which encoded the L526M, M550I, and L526M M550V drug resistance mutations were generated and used to examine the effects of these substitutions on viral sensitivity to lamivudine, penciclovir (the active form of famciclovir), and adefovir, three compounds of clinical importance. The following observations were made: (i) the L526M mutation confers resistance to penciclovir and partial resistance to lamivudine, (ii) the YMDD mutations M550I and L526M M550V confer high levels of resistance to lamivudine and penciclovir, and (iii) adefovir is active against each of these mutants. These findings are supported by the limited amount of clinical data currently available and confirm the utility of the HBV-baculovirus system as an in vitro tool for the molecular characterization of clinically significant HBV strains.

scholarly journals Inhibition of Hepatitis B Virus by AAV8-Derived CRISPR/SaCas9 Expressed From Liver-Specific Promoters

2021 ◽  
Vol 12 ◽  
Author(s):  
Kun Yan ◽  
Jiangpeng Feng ◽  
Xing Liu ◽  
Hongyun Wang ◽  
Qiaohong Li ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hbv Replication ◽  
Guide Rnas ◽  
Hbv Genotypes ◽  
Chronic Hepatitis B Virus ◽  
B Virus ◽  
Key Barrier

Curative therapies for chronic hepatitis B virus (HBV) infection remain a distant goal, and the persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is a key barrier that is hard to break through using the drugs currently approved for HBV treatment. Due to the accuracy, efficiency, and cost-effectiveness of genome editing, CRISPR/Cas technologies are being widely used for gene therapy and in antiviral strategies. Although CRISPR/Cas could possibly clear cccDNA, ensuring its safety is requirement for application. In our study, we analyzed the liver specificity of several promoters and constructed candidate promoters in the CRISPR/Staphylococcus aureus Cas9 (SaCas9) system combined with hepatotropic AAV8 (whereby AAV refers to adeno-associated virus) to verify the efficacy against HBV. The results revealed that the reconstructed CRISPR/SaCas9 system in which the original promoter replaced with a liver-specific promoter could still inhibit HBV replication both in vitro and in vivo. Three functional guide RNAs (gRNAs), T2, T3, and T6, which target the conserved regions of different HBV genotypes, demonstrated consistently better anti-HBV effects with different liver-specific promoters. Moreover, the three gRNAs inhibited the replication of HBV genotypes A, B, and C to varying degrees. Under the action of the EnhII-Pa1AT promoter and AAV8, the expression of SaCas9 was further decreased in other organs or tissues in comparison to liver. These results are helpful for clinical applications in liver by ensuring the effects of the CRISPR/Cas9 system remain restricted to liver and, thereby, reducing the probability of undesired and harmful effects through nonspecific targeting in other organs.

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.

Sign in / Sign up

Export Citation Format

Share Document