Kinetics of Hepadnavirus Loss from the Liver during Inhibition of Viral DNA Synthesis

Abstract

Hepadnaviruses replicate by reverse transcription, which takes place in the cytoplasm of the infected hepatocyte. Viral RNAs, including the pregenome, are transcribed from a covalently closed circular (ccc) viral DNA that is found in the nucleus. Inhibitors of the viral reverse transcriptase can block new DNA synthesis but have no direct effect on the up to 50 or more copies of cccDNA that maintain the infected state. Thus, during antiviral therapy, the rates of loss of cccDNA, infected hepatocytes (1 or more molecules of cccDNA), and replicating DNAs may be quite different. In the present study, we asked how these losses compared when woodchucks chronically infected with woodchuck hepatitis virus were treated with L-FMAU [1-(2-fluoro-5-methyl-␤-L-arabinofuranosyl) uracil], an inhibitor of viral DNA synthesis. Viremia was suppressed for at least 8 months, after which drug-resistant virus began replicating to high titers. In addition, replicating viral DNAs were virtually absent from the liver after 6 weeks of treatment. In contrast, cccDNA declined more slowly, consistent with a half-life of ϳ33 to 50 days. The loss of cccDNA was comparable to that expected from the estimated death rate of hepatocytes in these woodchucks, suggesting that death of infected cells was one of the major routes for elimination of cccDNA. However, the decline in the actual number of infected hepatocytes lagged behind the decline in cccDNA, so that the average cccDNA copy number in infected cells dropped during the early phase of therapy. This observation was consistent with the possibility that some fraction of cccDNA was distributed to daughter cells in those infected hepatocytes that passed through mitosis.

Immune selection during chronic hepadnavirus infection

“…There is no totally reliable measurement of hepatocyte turnover rates in the chronically infected liver, but at least some estimates put it in the range of 1-5% per day [11,12]. This contrasts with the levels of hepatocyte turnover in a healthy, uninfected liver, which are generally in the order of 0.01-0.1% per day (here we used 0.05%).…”

“…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].…”

“…cccDNA was assumed to survive mitosis, distributing to progeny hepatocytes in a binomial fashion. We considered liver turnover to be of the order of 1-5% of hepatocytes per day [11,12]. Figure 4a shows that if infected hepatocytes were killed at a rate of 1% per day, after 30 years, the frequency of HBeAg (-) HBV mutants would only rise about 100-fold, and hepatocytes containing only HBeAg (-) HBV would represent only about 1% of the total hepatocyte population.…”

“…In chronic infections, hepatocyte turnover is of the order of 1-5% per day or less [11,12]. Thus, in contrast to transient HBV infections, chronic infections appear to involve persistent immune attack on infected hepatocytes, which can cause substantial clinical disease, but is nonetheless inadequate to resolve the infection [13].…”

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.…”