Article

Detection of clonally expanded hepatocytes in chimpanzees with chronic hepatitis B virus infection.

Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Journal of Virology (Impact Factor: 5.08). 06/2009; 83(17):8396-408. DOI: 10.1128/JVI.00700-09
Source: PubMed

ABSTRACT During a hepadnavirus infection, viral DNA integrates at a low rate into random sites in the host DNA, producing unique virus-cell junctions detectable by inverse nested PCR (invPCR). These junctions serve as genetic markers of individual hepatocytes, providing a means to detect their subsequent proliferation into clones of two or more hepatocytes. A previous study suggested that the livers of 2.4-year-old woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus contained at least 100,000 clones of >1,000 hepatocytes (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. USA 102:1139-1144, 2005). However, possible correlations between sites of viral-DNA integration and clonal expansion could not be explored because the woodchuck genome has not yet been sequenced. In order to further investigate this issue, we looked for similar clonal expansion of hepatocytes in the livers of chimpanzees chronically infected with hepatitis B virus (HBV). Liver samples for invPCR were collected from eight chimpanzees chronically infected with HBV for at least 20 years. Fifty clones ranging in size from approximately 35 to 10,000 hepatocytes were detected using invPCR in 32 liver biopsy fragments (approximately 1 mg) containing, in total, approximately 3 x 10(7) liver cells. Based on searching the analogous human genome, integration sites were found on all chromosomes except Y, approximately 30% in known or predicted genes. However, no obvious association between the extent of clonal expansion and the integration site was apparent. This suggests that the integration site per se is not responsible for the outgrowth of large clones of hepatocytes.

0 Bookmarks
 · 
103 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: More than 350 million people worldwide are chronically infected with the human hepatitis B virus (HBV). Chronic HBV infections are associated with the development of hepatocellular carcinoma. While the mechanism of HBV-associated carcinoma remains undefined, it is thought to involve a combination of a continuous inflammatory response to HBV-infected hepatocytes and activities of HBV proteins such as the HBV X protein (HBx). HBx stimulates HBV replication; however, the mechanism by which HBx stimulates HBV replication remains incompletely understood. Studies performed with the woodchuck hepatitis virus (WHV) in woodchucks demonstrated that a C-terminally truncated mutant of the WHV X protein could not stimulate WHV replication. However, whether the C-terminus of HBx is important for HBx-stimulation of HBV replication is unclear. We have constructed C-terminal truncation mutants of HBx and have demonstrated that the C-terminus of HBx impacts HBx stability, HBx activation of transcription, and HBx stimulation of HBV replication. These observations highlight the impact of the HBx C-terminus on HBx activities and the importance of directly analyzing HBx expression and functions in HBV-associated tumors that contain chromosomal integrants of HBV with truncations of the HBx gene.
    Virus Research 10/2010; 155(1):231-9. · 2.75 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 = 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 07/2010; 52(1):16-24. · 12.00 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Chronic hepatitis B virus (HBV) infections are associated with persistent immune killing of infected hepatocytes. Hepatocytes constitute a largely self-renewing population. Thus, immune killing may exert selective pressure on the population, leading it to evolve in order to survive. A gradual course of hepatocyte evolution toward an HBV-resistant state is suggested by the substantial decline in the fraction of infected hepatocytes that occurs during the course of chronic infections. Consistent with hepatocyte evolution, clones of >1,000 hepatocytes develop postinfection in the noncirrhotic livers of chimpanzees chronically infected with HBV and of woodchucks infected with woodchuck hepatitis virus (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. U. S. A. 102:1139-1144, 2005; W. S. Mason et al., J. Virol. 83:8396-8408, 2009). The present study was carried out to determine (i) if extensive clonal expansion of hepatocytes also occurred in human HBV carriers, particularly in the noncirrhotic liver, and (ii) if clonal expansion included normal-appearing hepatocytes, not just hepatocytes that appear premalignant. Host DNA extracted from fragments of noncancerous liver, collected during surgical resection of hepatocellular carcinoma (HCC), was analyzed by inverse PCR for randomly integrated HBV DNA as a marker of expanding hepatocyte lineages. This analysis detected extensive clonal expansion of hepatocytes, as previously found in chronically infected chimpanzees and woodchucks. Tissue sections were stained with hematoxylin and eosin (H&E), and DNA was extracted from the adjacent section for inverse PCR to detect integrated HBV DNA. This analysis revealed that clonal expansion can occur among normal-appearing human hepatocytes.
    Journal of Virology 08/2010; 84(16):8308-15. · 5.08 Impact Factor

Full-text (2 Sources)

View
2 Downloads
Available from
Aug 10, 2014