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

Abstract

Chronic hepatitis B virus (HBV) infection is maintained by the presence of covalently closed circular DNA (cccDNA), the template of viral transcription and replication. In quiescent hepatocytes, cccDNA is a stable molecule that can persist throughout the hepatocyte lifespan. However, in chronic HBV infection, immunomediated cell injury and compensatory hepatocyte proliferation may favor cccDNA decline and selection of cccDNA-free cells. To investigate the impact of liver regeneration on cccDNA stability and activity in vivo, we used the urokinase-type plasminogen activator (uPA)/severe combined immunodeficiency (SCID) mouse model. Primary tupaia hepatocytes (PTHs) chronically infected with woolly monkey HBV (WM-HBV) were isolated from one highly viremic uPA/SCID chimeric mouse and transplanted into 20 uPA recipients. Expansion of transplanted PTHs and viral load changes were determined by real-time polymerase chain reaction and immunohistochemistry. Transplantation of WM-HBV infected hepatocytes led to an average of 3.8 PTH doublings within 80 days, 75% reduction of virion productivity (relaxed circular DNA/cccDNA), and lower expression levels of pregenomic RNA and hepatitis B core antigen. Remarkably, a median 2-log decline of cccDNA per cell determined during PTH proliferation was due to both dilution of the cccDNA pool among daughter cells and a 0.5-log loss of intrahepatic cccDNA loads (P 5 0.02). Intrahepatic viral DNA sequences persisting at the end of the study were mostly present as replicative intermediates and not as integrated virus. Conclusion: Cell division in the setting of liver regeneration and without administration of antiviral drugs induced strong destabilization of the cccDNA reservoir, resulting in cccDNA clearance in the great majority of chronically infected hepatocytes. (HEPATOLOGY 2010;52:16-24)

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

“…It should be noted that double-stranded linear DNA (dslDNA) was also present, since SpeI digestion resulted in a 1.2-kb band in addition to 3.2-kb species (lane 8). A heat resistance assay also confirmed the cccDNA form in protein-free DNA from liver biopsies when samples were heated from 75°C to 95°C ( Figure 3B, lanes 2-5), while PF-rcDNA from HepAD38 cells (lanes 6-9) and core particle DNA from a liver biopsy (lanes [11][12][13][14] were readily denatured. In addition, topoisomerase I treatment transformed the cccDNA into the relaxed form, as shown in Figure 3C (lanes 1 and 2), while PF-rcDNA from HepAD38 cells showed no mobility change (lanes 4 and 5).…”

“…The detailed analyses of the key steps required for the formation of HBV cccDNA were mostly cells constantly divide. Meanwhile, it is well recognized that hepatocyte proliferation markedly reduces intrahepatic cccDNA loads in vivo (14). Thus, this layer of difference may contribute to the low efficiency of cccDNA formation in cell lines.…”

Animal Models and the Molecular Biology of Hepadnavirus Infection

“…To address this distinction, Dandri et al (Lutgehetmann et al 2010) studied urokinase plasminogen activator-severe combined immunodeficiency disorder (uPA-SCID) mice (Sandgren et al 1991;Rhim et al 1995), in which the liver was partially repopulated with HBV-infected hepatocytes from tree shrews. A substantial loss of cccDNA was seen during expansion of the donor hepatocyte population, which appeared consistent with loss of cccDNA at mitosis (Lutgehetmann et al 2010), supporting the notion that cell division plays an important role in cccDNA loss during resolution of transient infections.…”

170 Ectopic Expression of Murine Cd47 Minimizes Macrophage Rejection of Human Hepatocyte Xenografts in Immunodeficient Mice

“…Humanized mice are either transgenic animals expressing human genes and/or immunodeficient mice engrafted with human cells or tissues (1). Immunodeficient mice repopulated with human hepatocytes have already proven useful for the study of hepatitis virus life cycles and new antiviral approaches (2,3) and various immunodeficient strains have emerged from different labs over time (4).…”