Half-Life of the Duck Hepatitis B Virus Covalently Closed Circular DNA Pool In Vivo following Inhibition of Viral Replication

Abstract

Covalently closed circular DNA (cccDNA) is a crucial intermediate in the replication of hepadnaviruses. We inhibited the replication of duck hepatitis B virus in congenitally infected ducks with a combination of lamivudine and a dideoxyguanosine prodrug. Inhibition of viral replication should prevent renewal of the cccDNA pool, and its decay was measured in liver biopsy samples collected over a 5-month period. In three ducks, the cccDNA pools declined exponentially, with half-lives ranging from 35 to 57 days. In two others, the pools declined exponentially for about 70 days but then stabilized at about 6 copies/diploid genome. The selection of drug-resistant virus mutants is an unlikely explanation for this unexpected stabilization of cccDNA levels. Liver sections stained for the cell division marker PCNA showed that animals in which cccDNA loss was continuous had significantly greater numbers of PCNA-positive nuclei than did those animals in which cccDNA levels had plateaued.The hepadnaviruses include the human pathogen Hepatitis B virus (HBV) and animal viruses such as Duck hepatitis B virus (DHBV) and Woodchuck hepatitis B virus (WHV), which are important models for studying hepadnavirus biology. These viruses are characterized by small, circular, partially doublestranded DNA genomes. The minus strand of the viral DNA is covalently linked to protein at its 5Ј end and contains a nick at its 3Ј end. The plus strand is only partially complete (for reviews, see references 10 and 21). A crucial event in the replication of these viruses is the conversion of this form of the viral genome into a covalently closed circular DNA (cccDNA) form within the nucleus of a newly infected hepatocyte (15,17,26). cccDNA is the template for viral transcripts encoding structural proteins and the viral polymerase. It is also the template for the pregenomic RNA which is the precursor of the viral genome. Early in infection, the pool of nuclear cccDNA is amplified to about 20 molecules/cell (13,14,24). This is accomplished when an intracellular circuit of pregenomic RNA is encapsidated and converted to viral DNA in the cytoplasm and subsequently enters the nucleus and converts to cccDNA (29). Once a pool of cccDNA is established, capsids containing newly replicated viral DNA are enveloped and exported from the cell rather than entering the nucleus. This change in the fate of nucleocapsids is thought to be caused by rising levels of the pre-S envelope protein resulting from an increasing pool of cccDNA template (22, 23).Attempts to cure hepadnavirus infections by treatment with antiviral drugs suggest that the cccDNA pool is very stable. If cccDNA turnover were rapid, then inhibitors of the viral reverse transcriptase would prevent replenishment of the cccDNA pool, leading to its elimination after a short course of treatment. However, human HBV carriers treated with lamivudine for 3 months usually relapse quickly when treatment is discontinued, indicating that cccDNA persists (5). If treatment is continued for 1 to 2 years, drug-resist...

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

“…[9][10][11] Notably, studies in the duck model showed that antiviral therapy with polymerase inhibitors induced a greater cccDNA reduction in animals displaying higher hepatocyte proliferation rates. 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.…”

“…Understanding molecular mechanisms affecting stability and activity of the cccDNA in vivo may reveal new therapeutic targets for the development of novel antiviral strategies able to enhance cccDNA clearance. 11,20 Although previous studies have noted that cccDNA turnover is slow even following administration of potent polymerase inhibitors, [5][6][7]12 little is known about the kinetics of cccDNA decay in the context of liver regeneration. WM-HBV-infected PTHs generated in uPA chimeric mice do not undergo hepatocellular changes, such as lipid droplet accumulation, nor do they show any sign of damage after longterm residence in immunosuppressive uPA mice, as has been reported for human hepatocytes.…”

Residual integrated viral DNA after hepadnavirus clearance by nucleoside analog therapy

“…focused primarily on a strategy of inhibiting viral DNA synthesis through the use of nucleoside analogs that are highly specific for the viral DNA polymerase (1)(2)(3)(4)(5)(6)(7)(8)(9). Treatment of patients with such inhibitors can be remarkably effective in reducing viremias to very low levels (2,3).…”

Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients

“…Most attempts for the quantitation of cccDNA have been made with liver biopsies from ducks or woodchucks. [11][12][13][14][15][16] Quantitation of cccDNA in human peripheral blood mononuclear cells and liver biopsies has been performed. [17][18][19][20] In these studies, primers spanning across the gap in the minus strand and corresponding to the variable region on the plus strand were used to amplify across noninterrupted cccDNA.…”