The cyclic adenosine monophosphate-response element (CRE)-transcription factor complex participates in the regulation of viral gene expression and pathologic processes caused by various viruses. The hepatitis B virus (HBV) enhancer I directs liver-specific transcription of viral genes and contains a CRE sequence (HBV-CRE); however, whether the HBV-CRE and CRE-binding protein (CREB) are required for the HBV life cycle remains to be determined. This study was designed to investigate the role of CREB in HBV replication and gene expression. Sequencecomparison analysis of 984 HBVs reported worldwide showed that the HBV-CRE sequence is highly conserved, indicating the possibility that it plays an important role in the HBV life cycle. The binding of CREB to the HBV-CRE site was markedly inhibited by oligonucleotides containing HBV-CRE and consensus CRE sequences in vitro and in vivo. The HBV promoter activity was demonstrated to be dependent upon the transactivation activity of CREB. Treatment with CRE decoy oligonucleotides reduced HBV promoter activity, and this was reversed by CREB overexpression. The levels of viral transcripts, DNA, and antigens were remarkably decreased in response to the overexpression of CREB mutants or treatment with the CRE decoy oligonucleotides, whereas enhancing CREB activity increased the levels of viral transcripts. In addition, introduction of a three-base mutation into the HBV-CRE led to a marked reduction in HBV messenger RNA synthesis. Conclusion: Taken together, our results demonstrate that both replication and gene expression of HBV require a functional CREB and HBV-CRE. We have also demonstrated that CRE decoy oligonucleotides and the overexpression of CREB mutants can effectively block the HBV life cycle, suggesting that interventions against CREB activity could provide a new avenue to treat HBV infection. (HEPATOLOGY 2008;48:361-373.) T he World Health Organization has reported that about 4 million people worldwide are acutely infected with hepatitis B virus (HBV) each year, and there are estimated to be 350 million chronic carriers worldwide. 1 The HBV is a major cause of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. [1][2][3] The agents that are currently available for the treatment of chronic HBV infection are interferon-␣ and nucleos(t)ide analogs, such as lamivudine (L-2Ј,3Ј-dideoxy-thiacytidene), adefovir dipivoxil, and entecavir. 2-5 However, there are some limitations to these therapeutics: interferon-␣ has adverse effects, and nucleos(t)ide derivatives, although safe and efficacious, can lead to the emergence of resistant mutants. [2][3][4][5] Furthermore, none of these therapeutic agents can effectively eradicate the virus. 4,5 Studies on the regulatory mechanisms of HBV gene expression and replication are, therefore, necessary for the development of promising antiviral medications. Interventions against HBV include novel nucleic acid-based approaches employing antisense RNA and DNA, 6-8 ribozymes, 9,10 and RNA interference. [11][12][13]

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