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Hepatitis B virus (hepadnavirus) infections are maintained by the presence of a small and regulated number of episomal viral genomes [covalently closed circular DNA (cccDNA)] in the nuclei of infected cells. Although a number of studies have measured the mean copy number of cccDNA molecules in hepadnaviral-infected cells, the distribution of individual copy numbers have not been reported. Using a PCR-based assay, we examined the number of cccDNA molecules of the duck hepatitis B virus in single nuclei isolated from the liver of a chronically infected duck over the course of 131 days of infection. Nuclei were isolated from frozen serial biopsies and individually deposited into PCR microplates by flow sorting. Each nucleus was assayed by nested PCR for cccDNA and for cellular IFN-α genes as an internal control. We found that 90% of the nuclei assayed contained between 1 and 17 cccDNA molecules, with the remaining 10% containing more (90% confidence), and that differences in the mean number of copies and distribution of copy numbers occurred within the same animal at different times postinfection. Overall, the data suggest (i) that the number of cccDNA molecules per cell may fluctuate over time, and (ii) that, according to these fluctuations, a substantial fraction of cells may contain only one or a few copies. We infer from the results that infected hepatocytes express virus at different levels and that during cell division it is possible to segregate cells containing no cccDNA. (Copyright 2003, National Academy of Sciences, U.S.A.)
Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver. , , , , , . Proc Natl Acad Sci U S A 2003; 100: 12372-12377. (Reprinted by permission from National Academy of Sciences, U.S.A.)
Hans Christian Spangenberg M.D.*, Robert Thimme M.D.*, Hubert E. Blum M.D.*, * Department of Medicine II, University Hospital Freiburg, Freiburg, Germany.
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Hepatitis B virus (HBV), the prototype member of the Hepadnaviridae family, is a small noncytopathic, hepatotropic virus with more than 350 million infected individuals worldwide that causes acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The virus has a partially double-stranded relaxed circular DNA genome that is converted into a covalently closed circular DNA (cccDNA) episome in the nucleus of infected cells.1, 2 The cccDNA serves as the transcriptional template for four species of viral RNAs that code for the viral structural and nonstructural proteins, including the reverse transcriptase / DNA polymerase. The viral pregenomic RNA is encapsidated into core particles and serves as template for the reverse transcription into minus-strand DNA followed by plus-strand DNA synthesis to yield partially double-stranded relaxed circular DNA. Core particles containing relaxed circular DNA are either enveloped and exported as virions or they deliver their DNA content to the nucleus, thereby amplifying the pool of cccDNA molecules.3–5 The factors regulating cccDNA content of infected cells as well as the kinetics of cccDNA during acute self-limited and chronic HBV infection are not fully understood.
Zhang et al.6 analyzed the cccDNA content at the single-cell level in the well established duck hepatitis B virus (DHBV) model of HBV infection to elucidate the natural course of the cccDNA species during infection. After DHBV infection of a duckling, liver biopsies were obtained at various time points up to 131 days after infection. Liver cell nuclei were isolated and sorted by a cytometer and a high-speed cell sorter to yield a single nucleus per well, followed by quantitative polymerase chain reaction to measure the cccDNA and interferon-α copy numbers as an internal control. Ninety percent of hepatocytes contained 1 to 17 cccDNA molecules, while 10% contained more. Interestingly, in the DHBV-infected animal the mean copy number of cccDNA ranged between 2.9 and 8.6 copies per cell during the observation period of 131 days, with lowest copy numbers and viremia levels at day 88. The distribution of cccDNA varied significantly within a liver biopsy with up to 10 to 36 copies per cell. There was, however, always a significant fraction of cells containing only 1 or few cccDNA molecules. These findings suggest that the single-cell cccDNA content might be influenced by various mechanisms such as liver growth, regeneration, and inflammation during chronic infection. While this study was not designed to examine these factors, studies in chronically infected ducks treated with reverse transcriptase inhibitors,7 studies in chronically woodchuck hepatitis virus–infected animals treated with inhibitors of viral replication,4, 8 and in woodchuck hepatitis virus–infected woodchucks with acute, self-limited infection5, 9 indicate that cccDNA elimination depends on the death and turnover of infected hepatocytes. Noncytolytic immune-mediated mechanisms that inhibit HBV replication have been well described in HBV transgenic mice10–12 and HBV infected chimpanzees.13, 14 In the HBV transgenic mouse model, virus-specific CD8+ T cells and other stimuli that trigger the production of interferons in the liver lead to an inhibition of viral replication by eliminating the pregenomic RNA–containing capsids from the hepatocyte cytoplasm.15, 16 Since transgenic mice do not produce cccDNA, the mechanisms responsible for the elimination of cccDNA cannot be determined in this model.
A recent study that analyzed the expansion and contraction of the HBV transcriptional template in infected chimpanzees indicated that cccDNA and HBV replicative intermediates are controlled by noncytolytic and cytolytic mechanisms during immune-mediated clearance of HBV infection.17 During the early phase of infection, it appeared that HBV replicative intermediates and cccDNA were controlled by noncytolytic mechanisms. Replicative intermediates, however, were more sensitive to the noncytolytic immune-mediated inhibition than cccDNA. In the later phase of infection, cytolytic mechanisms led to final viral elimination. Interestingly, despite the destruction and regeneration of hepatocytes during the final stages of acute hepatitis, the cccDNA persisted indefinitely at very low levels after resolution of infection, possibly explaining lifelong immune responses to HBV despite clinical resolution of HBV infection.18, 19
Though the chimpanzee study was not designed to analyze the cccDNA at a single-cell level and the study by Zhang et al.6 was not performed to elucidate the mechanisms responsible for changes in cccDNA, these studies allow new insights in the natural course of cccDNA molecules in acute self-limited and chronic HBV infection.
Zhang et al.6 elegantly measured the cccDNA content in single nuclei. Their most important finding is that cccDNA molecules per cell vary broadly in chronic HBV infection and that hepadnaviruses can support stable infection with small numbers of cccDNA molecules. As the authors speculate, it is even possible that during cell division some infected cells are segregated that contain no cccDNA because it was lost through cell division or some other process.
Based on the technology used, the exclusive hepatocyte origin of nuclei is difficult to prove. Therefore, it is possible that especially nuclei with low cccDNA or negative for this viral DNA species may be derived from nonhepatocytes present in the liver biopsy. Apart from analyzing the host factors determining the cccDNA copy number in individual DHBV-infected hepatocytes, it would be most interesting to study early steps of hepadnaviral infection with the methology used. Such analyses should allow to determine the spread of DHBV after infection from, possibly, very few infected liver cells with high or very high cccDNA copy numbers to more liver cells with lower or no cccDNA copies in the course of infection.
Taken together, the important and interesting findings of Zhang et al.6 show for the first time that the number of cccDNA molecules per cell does not seem to be crucial to the natural course of HBV infection and that the relatively low numbers of cccDNA are sufficient to maintain persistent infection. Thus, permanently clear or control HBV infection, antiviral strategies should not only downregulate or eradicate HBV DNA species but also should induce or enhance the immune responses against HBV.