Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

ABSTRACTHepatitis B virus (HBV)-associated chronic liver diseases are treated with nucleoside analogs that target the virus polymerase. While these analogs are potent, drugs are needed to target other virus-encoded gene products to better block the virus replication cycle and chronic liver disease. This work further characterized GLS4 and compared it to the related BAY 41-4109, both of which trigger aberrant HBV core particle assembly, where the virus replication cycle occurs. This was done in HepAD38 cells, which replicate HBV to high levels.In vitro, GLS4 was significantly less toxic for primary human hepatocytes (P< 0.01 up to 100 μM), inhibited virus accumulation in the supernantant of HepAD38 cells (P< 0.02 up to 100 nM), inhibited HBV replicative forms in the liver with a significantly lower 50% effective concentration (EC50) (P< 0.02), and more strongly inhibited core gene expression (P< 0.001 at 100 to 200 nM) compared to BAY 41-4109.In vivocharacterization was performed in nude mice inoculated with HepAD38 cells, which grew out as tumors, resulting in viremia. Treatment of mice with GLS4 and BAY 41-4109 showed strong and sustained suppression of virus DNA to about the same extents both during and after treatment. Both drugs reduced the levels of intracellular core antigen in the tumors. Alanine aminotransferase levels were normal. Tumor and total body weights were not affected by treatment. Thus, GLS4 was as potent as the prototype, BAY 41-4109, and was superior to lamivudine, in that there was little virus relapse after the end of treatment and no indication of toxicity.

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Inhibition of Hepatitis B Virus Replication by the Interferon-Inducible MxA Protein

Journal of Virology ◽

10.1128/jvi.75.6.2684-2691.2001 ◽

2001 ◽

Vol 75 (6) ◽

pp. 2684-2691 ◽

Cited By ~ 115

Author(s):

Emmanuel Gordien ◽

Olivier Rosmorduc ◽

Cécile Peltekian ◽

Florianne Garreau ◽

Christian Bréchot ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Antiviral Activity ◽

Posttranscriptional Regulation ◽

Antiviral Effect ◽

Complete Disappearance ◽

Reporter System ◽

B Virus ◽

Host Defense System ◽

Nucleocytoplasmic Export

ABSTRACT Human MxA is an alpha/beta interferon-inducible intracytoplasmic protein that mediates antiviral activity against several RNA viruses. We had previously shown that overexpression of the hepatitis B virus (HBV) capsid led to selective downregulation of MxA gene expression, suggesting a mechanism by which the virus escapes from the host defense system (O. Rosmorduc, H. Sirma, P. Soussan, E. Gordien, P. Lebon, M. Horisberger, C. Brechot and D. Kremsdorf, J. Gen. Virol. 80:1253–1262, 1999). In the present study, we investigated the antiviral activity of MxA protein against HBV. MxA-expressing HuH7 clones were established and transiently transfected with HBV, and viral replication was then studied. Viral protein secretion was profoundly reduced in MxA-expressing clones by 80% for HBV surface antigen (HBsAg) and 70% for HBV e antigen (HBeAg). The levels of intracytoplasmic HBsAg and HBeAg were reduced by about 80 and 50% in the two MxA-positive clones tested. A nearly complete disappearance of HBV DNA replicative intermediates was observed in MxA-expressing clones. Although the expression of total viral RNAs was not modified, two- to fourfold reductions in HBV cytoplasmic RNAs were found in MxA-expressing clones. This suggests the inhibition of HBV replication at a posttranscriptional level. Indeed, using the well-characterized posttranscriptional regulation element (PRE) reporter system, we were able to demonstrate a marked reduction (three- to eightfold) in the nucleocytoplasmic export of unspliced RNA in MxA-expressing clones. In addition, MxA protein did not interact with HBV nucleocapsid or interfere with HBV nucleocapsid formation. Our results show an antiviral effect of MxA protein on a DNA virus for the first time. MxA protein acts, at least in part, by inhibiting the nucleocytoplasmic export of viral mRNA via the PRE sequence.

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