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Differential Stability of Nuclear and Cytoplasmic Hepatitis B Virus Replication Intermediates in Response to IFNα/ß

Hepatitis B virus (HBV), an enveloped virus that infects the liver of humans and other primates, causes both acute and chronic disease. According to estimates, worldwide approximately 300 million persons are chronic HBV carriers.

In infected hepatocytes, the 3.2-kb DNA genome of HBV is transcribed by the cellular RNA polymerase II to generate the 3.5-, 2.4-, 2.1-, and 0.7-kb viral RNAs. These transcripts encode the nucleocapsid polypeptides, the large surface-antigen polypeptide, the middle and major surface-antigen polypeptides, and the X-gene polypeptide, respectively. In addition, the 3.5-kb pregenomic RNA encodes the viral polymerase and is reverse transcribed by this polymerase within the viral nucleocapsid to produce the 3.2-kb viral genomic DNA. The mature nucleocapsids containing viral genomic DNA can only be secreted from the cell in virus particles by associating with surface-antigen polypeptides within the membrane of the endoplasmic reticulum. Virus buds into the lumen of the endoplasmic reticulum and is transported out of the cell through the Golgi apparatus. The ability of the mature nucleocapsid to form virus particles depends on the correct level of synthesis of the surface-antigen polypeptides. If no synthesis of surface-antigen polypeptide occurs, the mature nucleocapsids transport the viral genome back into the nucleus. There the partially double-stranded viral genome is converted into covalently closed circular (CCC) DNA that represents the viral transcriptional template. Therefore, the level of transcription of the 2.4- and 2.1-kb viral RNAs, in addition to the 3.5-kb pregenomic RNA, influences viral replication in general and nuclear accumulation of HBV CCC DNA in particular.

HBV transgenic mice that lack hepatocyte nuclear factor 1α synthesize nuclear HBV CCC DNA, a characteristic that allows us to compare the stability of this replication intermediate (the CCC DNA) with cytoplasmic encapsidated viral replication intermediates. Treatment of these HBV transgenic mice with various immunologic stimuli such as polyinosinic polycytidylic acid results in the modulation of viral replication. Treatment with polyinosinic polycytidylic acid, a double-stranded RNA, stimulates cells to synthesize various cytokines, primarily IFN-α/ß, which initiates cellular processes to limit viral infections. We found that treatment with polyinosinic polycytidylic acid reduced cytoplasmic synthesis of viral replication intermediates more than 10-fold but had no effect on nuclear levels of HBV CCC DNA. This observation suggests that nuclear HBV CCC DNA is stable in the liver in contrast to the instability of encapsidated replication intermediates in the cytoplasm.

The stability of HBV CCC DNA is a critical issue in resolving chronic HBV infections. Although current treatments reduce viral loads in patients with chronic HBV infections, the infection is often not resolved. Presumably, this result is due to the persistence of HBV CCC DNA in the infected liver. Additionally, HBV CCC DNA is the viral replication intermediate responsible for the integration of HBV DNA into the host chromosomal DNA associated with HBV-mediated hepatocellular carcinoma. This finding suggests that physiologic or therapeutic stimuli that downregulate cytoplasmic synthesis of viral replication intermediates but do not modulate nuclear HBV CCC DNA, such as IFN-α/ß, most likely will not lead to resolution of chronic HBV infections or reduce the rates of HBV-induced hepatocellular carcinoma. Therefore, alternative antiviral therapies aimed at eliminating HBV CCC DNA are required. HBV transgenic mice that lack the gene for hepatocyte nuclear factor 1α are a useful model for examining the effect of new antiviral therapies on the synthesis and stability of HBV CCC DNA.

Publications

Tang, H., McLachlan, A. A pregenomic RNA sequence adjacent to DR1 and complementary to epsilon determines hepatitis B virus replication efficiency. Virology 303:199, 2002.