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The non-structural X protein, HBx, of hepatitis B virus (HBV) is assumed to play an important role in HBV replication. Woodchuck hepatitis virus X protein is indispensable for virus replication, but the duck hepatitis B virus X protein is not. In this study, we investigated whether the HBx protein is indispensable for HBV replication in vivo using human hepatocyte chimeric mice. HBx-deficient (HBx-def) HBV was generated in HepG2 cells by transfection with an overlength HBV genome. Human hepatocyte chimeric mice were infected with HBx-def HBV with or without hepatic HBx expression by hydrodynamic injection of HBx expression plasmids. Serum virus levels and HBV sequences were determined with mice sera. The generated HBx-def HBV peaked in the sucrose density gradient at points equivalent to the generated HBV wild type and the virus in a patient's serum. HBx-def HBV-injected mice developed measurable viraemia only in continuously HBx-expressed liver. HBV DNA in the mouse serum increased up to 9 log(10) copies ml(-1) and the viraemia persisted for more than 2 months. Strikingly, all revertant viruses had nucleotide substitutions that enabled the virus to produce the HBx protein. It was concluded that the HBx protein is indispensable for HBV replication and could be a target for antiviral therapy.

HBx protein is indispensable for development of viraemia in human hepatocyte chimeric mice