We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication.

AT-61, a member of a novel class of phenylpropenamide derivatives, was found to be a highly selective and potent inhibitor of human hepatitis B virus (HBV) replication in four different human hepatoblastoma cell lines which support the replication of HBV (i.e., HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cells). This compound was equally effective at inhibiting both the formation of intracellular immature core particles and the release of extracellular virions, with 50% effective concentrations ranging from 0.6 to 5.7 μM. AT-61 (27 μM) was able to reduce the amount of HBV covalently closed circular DNA found in the nuclei of HepAD38 cells by >99%. AT-61 at concentrations of >27 μM had little effect on the amount of viral RNA found within the cytoplasms of induced HepAD38 cells but reduced the number of immature virions which contained pregenomic RNA by >99%. The potency of AT-61 was not affected by one of the mutations responsible for (−)-β-l-2′,3′-dideoxy-3′ thiacytidine (3TC) resistance in HBV, and AT-61 acted synergistic with 3TC to inhibit HBV replication. AT-61 (81 μM) was not cytotoxic or antiproliferative to several cell lines and had no antiviral effect on woodchuck or duck HBV, human immunodeficiency virus type 1, herpes simplex virus type 1, vesicular stomatitis virus, or Newcastle disease virus. Therefore, we concluded that the antiviral activity of AT-61 is specific for HBV replication and most likely occurs at one of the steps between the synthesis of viral RNA and the packaging of pregenomic RNA into immature core particles.

No abstract

AT-61, a member of a novel class of phenylpropenamide derivatives, was found to be a highly selective and potent inhibitor of human hepatitis B virus (HBV) replication in four different human hepatoblastoma cell lines which support the replication of HBV (i.e., HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cells). This compound was equally effective at inhibiting both the formation of intracellular immature core particles and the release of extracellular virions, with 50% effective concentrations ranging from 0.6 to 5.7 M. AT-61 (27 M) was able to reduce the amount of HBV covalently closed circular DNA found in the nuclei of HepAD38 cells by >99%. AT-61 at concentrations of >27 M had little effect on the amount of viral RNA found within the cytoplasms of induced HepAD38 cells but reduced the number of immature virions which contained pregenomic RNA by >99%. The potency of AT-61 was not affected by one of the mutations responsible for (؊)-␤-L-2,3-dideoxy-3 thiacytidine (3TC) resistance in HBV, and AT-61 acted synergistic with 3TC to inhibit HBV replication. AT-61 (81 M) was not cytotoxic or antiproliferative to several cell lines and had no antiviral effect on woodchuck or duck HBV, human immunodeficiency virus type 1, herpes simplex virus type 1, vesicular stomatitis virus, or Newcastle disease virus. Therefore, we concluded that the antiviral activity of AT-61 is specific for HBV replication and most likely occurs at one of the steps between the synthesis of viral RNA and the packaging of pregenomic RNA into immature core particles.

The cytosine analog 2′-deoxy-3′-thiacytidine (3TC) has been shown to be an effective treatment for chronic hepatitis B virus (HBV) infection. However, several liver transplant patients who were undergoing treatment with 3TC for HBV infection experienced a breakthrough of virus while on 3TC. The predominant virus found in these patients’ sera contained either a valine or isoleucine for the methionine in the highly conserved YMDD nucleotide binding site in the HBV polymerase. To determine the biological relevance of the Met-to-Val substitution, we mutated a plasmid that contained a cDNA copy of the HBV pregenomic RNA such that when virus replication occurred during transient transfection of HepG2 cells, an M539V polymerase variant was produced. We found that in transiently transfected cells, this variant was approximately 330-fold less sensitive to the antiviral effects of 3TC and produced 7-fold less viral DNA than the wild type.