P.028 No amplification of the hepatitis B virus covalently closed circular DNA after in vitro infection of the human hepatoma HepaRG cells

ABSTRACTHepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in viral infection and persistence and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment. Therefore, there is an urgent need for the development of novel therapeutic agents that directly target cccDNA formation and maintenance. By employing an innovative cell-based cccDNA assay in which secreted HBV e antigen is a cccDNA-dependent surrogate, we screened an in-house small-molecule library consisting of 85,000 drug-like compounds. Two structurally related disubstituted sulfonamides (DSS), termed CCC-0975 and CCC-0346, emerged and were confirmed as inhibitors of cccDNA production, with low micromolar 50% effective concentrations (EC50s) in cell culture. Further mechanistic studies demonstrated that DSS compound treatment neither directly inhibited HBV DNA replication in cell culture nor reduced viral polymerase activity in thein vitroendogenous polymerase assay but synchronously reduced the levels of HBV cccDNA and its putative precursor, deproteinized relaxed circular DNA (DP-rcDNA). However, DSS compounds did not promote the intracellular decay of HBV DP-rcDNA and cccDNA, suggesting that the compounds interfere primarily with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck HBV-infected primary duck hepatocytes. This is the first attempt, to our knowledge, to identify small molecules that target cccDNA formation, and DSS compounds thus potentially serve as proof-of-concept drug candidates for development into therapeutics to eliminate cccDNA from chronic HBV infection.

Download Full-text

Inhibitory Effect of Adefovir on Viral DNA Synthesis and Covalently Closed Circular DNA Formation in Duck Hepatitis B Virus-Infected Hepatocytes In Vivo and In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.2.425-433.2002 ◽

2002 ◽

Vol 46 (2) ◽

pp. 425-433 ◽

Cited By ~ 64

Author(s):

Julien Delmas ◽

Olivier Schorr ◽

Catherine Jamard ◽

Craig Gibbs ◽

Christian Trépo ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Dna Synthesis ◽

Dna Amplification ◽

Viral Dna ◽

Circular Dna ◽

Covalently Closed Circular Dna ◽

B Virus

ABSTRACT The elimination of viral covalently closed circular DNA (CCC DNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with adefovir, a new acyclic nucleoside phosphonate, the prodrug of which, adefovir dipivoxil, is in clinical evaluation, is able to suppress viral CCC DNA both in vitro and in vivo using the duck HBV (DHBV) model. First, the effect of adefovir on viral CCC DNA synthesis was examined with primary cultures of DHBV-infected fetal hepatocytes. Adefovir was administered for six consecutive days starting one day before or four days after DHBV inoculation. Dose-dependent inhibition of both virion release in culture supernatants and synthesis of intracellular viral DNA was observed. Although CCC DNA amplification was inhibited by adefovir, CCC DNA was not eliminated by antiviral treatment and the de novo formation of CCC DNA was not prevented by pretreatment of the cells. Next, preventive treatment of experimentally infected ducklings with lamivudine or adefovir revealed that both efficiently suppressed viremia and intrahepatic DNA. However, persistence of viral DNA even when detectable only by PCR was associated with a recurrence of viral replication following drug withdrawal. Taken together, our results demonstrate that adefovir is a potent inhibitor of DHBV replication that inhibits CCC DNA amplification but does not effectively prevent the formation of CCC DNA from incoming viral genomes.

Download Full-text

n-Butyrate, a cell cycle blocker, inhibits early amplification of duck hepatitis B virus covalently closed circular DNA after in vitro infection of duck hepatocytes.

Journal of Virology ◽

10.1128/jvi.70.5.2691-2696.1996 ◽

1996 ◽

Vol 70 (5) ◽

pp. 2691-2696 ◽

Cited By ~ 14

Author(s):

F Turin ◽

C Borel ◽

M Benchaib ◽

A Kay ◽

C Jamard ◽

...

Keyword(s):

Cell Cycle ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Duck Hepatitis B Virus ◽

Circular Dna ◽

Covalently Closed Circular Dna ◽

Duck Hepatitis ◽

B Virus ◽

A Cell

Download Full-text

T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

Journal of Virology ◽

10.1128/jvi.01117-18 ◽

2018 ◽

Vol 92 (23) ◽

Cited By ~ 11

Author(s):

Bingqian Qu ◽

Yi Ni ◽

Florian A. Lempp ◽

Florian W. R. Vondran ◽

Stephan Urban

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Genomic Dna ◽

Circular Dna ◽

Covalently Closed Circular Dna ◽

Copy Numbers ◽

B Virus ◽

Replicative Intermediates ◽

Reliable Quantification

ABSTRACTChronic infection with the human hepatitis B virus (HBV) is a major health problem. Virus persistence requires the establishment and maintenance of covalently closed circular DNA (cccDNA), the episomal virus template in the nucleus of infected hepatocytes. Compared to replicative DNA intermediates (relaxed circular DNA [rcDNA]), copy numbers of cccDNA in infected hepatocytes are low. Accordingly, accurate analyses of cccDNA require enrichment of nuclear fractions and Southern blotting or selective quantitative PCR (qPCR) methods allowing discrimination of cccDNA and rcDNA. In this report, we analyzed cccDNA-specific primer pairs for their ability to amplify cccDNA selectively. Using mixtures of defined forms of HBV and genomic DNA, we determined the potential of different nucleases for targeted digestion of the open/relaxed circular DNA forms in the absence and presence of genomic DNA without affecting cccDNA. We found that the combination of T5 exonuclease with a primer set amplifying an approximately 1-kb fragment permits reliable quantification of cccDNA without the requirement of prior nucleus enrichment or Hirt extraction. We tested this method in four differentin vitroinfection systems and quantified cccDNA copy numbers at increasing multiplicities of inoculated genome equivalents. We further analyzed the kinetics of cccDNA formation and the effect of drugs (interferon, entry inhibitors, and capsid inhibitors) on cccDNA. Our method allows reliable cccDNA quantification at early stages of infection in the presence of a high excess of input virus and replicative intermediates and is thereby suitable for drug screening and investigation of cccDNA formation and maintenance.IMPORTANCEcccDNA elimination is a major goal in future curative regimens for chronic HBV patients. However, PCR-based assays for cccDNA quantification show a principally constrained specificity when high levels of input virus or replicative intermediates are present. Here, we characterized T5 exonuclease as a suitable enzyme for medium-throughputin vitroassays that preserves cccDNA but efficiently removes rcDNA prior to PCR-based quantification. We compared T5 exonuclease with the previously described exonuclease III and showed that both nucleases are suitable for reliable quantification of cccDNA by PCR. We substantiated the applicability of our method through examination of early cccDNA formation and stable accumulation in severalin vitroinfection models and analyzed cccDNA stability after administration of anti-HBV drugs. Our results support the use of T5 exonuclease for fast and convenient rcDNA removal, especially for early cccDNA quantification and rapid drug testing inin vitrostudies.

Download Full-text