Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver

Abstract

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-␣ 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.

In vivo proliferation of hepadnavirus-infected hepatocytes induces loss of covalently closed circular DNA in mice

“…9,10,27,28 In our experimental setting, virtually all PTHs engrafting the murine livers stained HBcAg-positive, and it is unlikely that expansion of PTHs occurred predominantly in uninfected cells, because in this case a significant intrahepatic cccDNA loss would not have occurred. Thus, in line with reports indicating that cccDNA loss is not only achieved through hepatocyte death, our findings provide the first direct evidence that regeneration of infected hepatocytes occurring without cell killing (Supporting Information) and in the absence of the adaptive immune response and polymerase inhibitors blocking new rounds of infection, induces drastic destabilization and significant clearance of the cccDNA pool in vivo.…”

“…However, it has been proposed that cell division may favor dilution of the cccDNA, so that cccDNA-free cells can be generated while infected cells are forced to divide to compensate for the immunomediated loss of other infected cells. [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.…”

Immune selection during chronic hepadnavirus infection

“…Attempts to address some of these issues have already been made in animal models of HBV infection. For instance, Zhang and Summers [62] found that competition between DHBV strains with different replication rates essentially stops once the liver is fully infected, suggesting that superinfection is probably inefficient, a conclusion that could now be further tested through analysis of the cccDNA content of individual nuclei [57].…”

“…The process is likely to be inefficient in as much as excessive accumulation of cccDNA does not seem to occur, most copy number estimates of cccDNA being in the range of 5-50 per hepatocyte. A study of single nuclei from DHBV-infected ducks also suggested that cccDNA copy numbers remain low [57], unlike what might be anticipated if the hepatocytes were being constantly superinfected. Hepatitis delta virus (HDV) has the HBV envelope, and its ability to superinfect the chronically HBV-infected liver might be taken as evidence that superinfection is an efficient process.…”

Serological and molecular epidemiological outcomes after two decades of universal infant hepatitis B virus (HBV) vaccination in Nunavut, Canada

“…The newly synthesized viral genome may have another fate and be transported back into the nucleus to restore the cccDNA pool [50]. Herein lies the difficulty in curing chronic infection, as it is extremely difficult to eliminate cccDNA pools in host hepatocytes' nuclei.…”