Increased hepatocyte turnover and inhibition of woodchuck hepatitis B virus replication by adefovir In vitro do not lead to reduction of the closed circular DNA

Abstract

The aim of this study was to evaluate the inhibitory effect of the nucleotide analogue adefovir on woodchuck hepatitis B virus (WHV) replication and, in particular, to determine whether the pool of covalently closed circular DNA (cccDNA) could be reduced by adefovir treatment in primary cultures of woodchuck hepatocytes isolated from a chronic carrier. Strong reduction of WHV-DNA synthesis (90%) and secretion (up to 98%) was observed with all 3 doses of adefovir used (1, 10, and 100 mol/L), whereas in the absence of the drug, high amounts of viral particles were continuously secreted in the culture medium until the end of the study (27 days). Secretion of envelope proteins and viral RNA levels remained constant both in the adefovirtreated and -untreated cultures for the entire course of the study. Intracellular core protein levels declined by approximately 50% in all the cultures, independent of adefovir treatment. There was no indication of cccDNA loss in the adefovir-treated hepatocyte cultures even when cell turnover was induced for 14 days by the addition of epidermal growth factor (EGF) to the culture medium. Our data show that adefovir has a very strong inhibitory effect on WHV-DNA synthesis in chronically infected primary hepatocyte cultures and indicate that cccDNA is a very stable molecule that appears to be efficiently transmitted to the dividing hepatocytes. (HEPATOLOGY 2000;32:139-146.)Persistent infection with hepatitis B virus (HBV) continues to be a major health problem as it is associated with various degrees of liver damage, cirrhosis, and hepatocellular carcinoma. Despite the existence of an effective vaccine, more than 350 million people are infected worldwide. 1,2 Current treatment regimens primarily rely on interferon alfa therapy. However, interferon treatment is associated with a variety of side effects and has been successful in only approximately one third of the patients, being very poorly effective in individuals with perinatally acquired HBV infection. In recent years, a variety of new anti-HBV agents have been developed, and these are currently evaluated in clinical trials. 3,4 Because HBV replication proceeds through reverse transcription, treatment with inhibitors of HBV reverse transcriptase is a very attractive option.During hepadnavirus infection, the relaxed circular partially double stranded DNA genome is transported to the nucleus of the hepatocyte, where it is converted into a covalently closed circular molecule, named cccDNA, which serves as template for the transcription of all viral RNAs. 5 The synthesis of the new relaxed circular viral DNA genome is mediated by the viral P-protein, which has reverse transcriptase and DNA-dependent DNA polymerase activity. Synthesis of the viral DNA takes place in the cytoplasm of the hepatocytes within immature nucleocapsids. 6 To establish a sufficient pool of nuclear cccDNA, some of the newly synthesized viral DNAs need to be retransported to the nucleus of the infected hepatocyte. 7 Therefore, the replicative forms of viral DNA are a...

Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B

“…4,5 Woodchuck studies 6,7 and recent clinical trials 8 demonstrated that significant reduction in the cccDNA pool was achieved only after long-term antiviral therapy. As HBV polymerase inhibitors do not have a direct effect on cccDNA formation, the decreased intrahepatic cccDNA levels are supposed to be derived from the lack of sufficient recycling of viral nucleocapsids to the nucleus because of the strong inhibition of viral DNA synthesis in the cytoplasm and less incoming viruses from the blood.…”

Immune selection during chronic hepadnavirus infection

“…Thus, it seems that either cccDNA is not lost at mitosis or the mutant does not replicate any more efficiently than the wild-type virus. Again, it is not known for certain if cccDNA survives mitosis, though some published evidence suggests this is the case [12,55].…”

In Vivo Proliferation of Hepadnavirus-Infected Hepatocytes Induces Loss of Covalently Closed Circular DNA in Mice

“…1 Due to this peculiar replication mechanism, cccDNA is not a direct target of current antiviral therapies. Experimental evidence from a woodchuck model and clinical trials shows that in the presence of polymerase inhibitors, the cccDNA pool persisted even when viral production was strongly reduced 4,5 and that long-term antiviral therapy is needed to achieve significant decrease of cccDNA levels. [6][7][8] Considering that cccDNA is very stable in the absence of cell division, 5 the development of resistant mutants escaping antiviral therapy may eventually occur.…”

“…12 A cccDNA decrease was also observed in hepatocytes chronically infected with woodchuck hepatitis virus when cell turnover was induced in vitro by addition of cellular growth factors and viral replication was suppressed by adefovir treatment. 4 Furthermore, identification of cccDNA-free woodchuck hepatocytes containing traces of the infection in form of viral integrations indicated that cccDNA clearance may occur without killing the infected cells. 9 Due to the narrow host range of HBV and limited availability of infection models, little is known about the impact of liver regeneration on cccDNA stability.…”