Hepatitis B virus (HBV) is a small DNA virus with a high liver tropism (for a review, see reference 11
). This virus contains a 3.2-kb circular, partially double-stranded DNA genome. Upon infection of hepatocytes, this genomic DNA is transported into the nucleus, where it is repaired to form a covalently closed circular DNA (cccDNA) molecule. This cccDNA then serves as the template for the transcription of HBV RNAs. The HBV genome contains four promoters named the pre-S1, S, core, and X promoters, which direct transcription of the pre-S1 protein mRNA and the pre-S2 protein, the major S protein mRNAs, the precore protein and the core protein mRNAs, and the X protein mRNA, respectively. Pre-S1, pre-S2, and major S proteins, which are collectively called surface antigens, are the viral envelope proteins. The precore protein is the precursor of the e antigen found in the serum of HBV patients, and the core protein is the viral capsid protein. The X protein is a transcriptional transactivator that can regulate cellular signaling pathways and modify the DNA binding activities of transcription factors (37
). The core protein mRNA also encodes the viral DNA polymerase, which is also a reverse transcriptase. In addition to these four promoters, the HBV genome also contains two enhancer elements, termed ENI and ENII. Both enhancers display liver-specific activities and are recognized by liver-enriched transcription factors. ENII is located in the upstream regulatory region of the core promoter and is also known as the core upstream regulatory sequence (38
After its translation, the core protein packages its own mRNA as well as the viral polymerase to form the core particle. The core mRNA, which is also known as the pregenomic RNA, is then reverse transcribed to form the circular, partially double-stranded HBV DNA genome. The core particle then interacts with the surface antigens on the membrane of the endoplasmic reticulum to form the progeny virions, which are then secreted from the infected cell (11
The replication of HBV can be affected by a variety of factors, including hormones, growth factors, and cytokines. For example, glucagon interferes with the replication of the related duck HBV (14
); transforming growth factor beta suppresses the replication of duck HBV; epidermal growth factor, transforming growth factor alpha, and hepatocyte growth factor stimulate the accumulation of duck HBV cccDNA in the nuclei of infected cells (5
); and interferons, tumor necrosis factor alpha, and insulin suppress the replication of HBV (12
). In addition, the cell cycle has also been shown to affect HBV replication: the replication of HBV is enhanced in cells arrested in the G1
phase and suppressed if cells enter the S phase (29
), and cccDNA accumulation appears to increase in the G1
). In spite of these observations, the molecular mechanisms by which external factors or the cell cycle affects HBV replication remain largely unknown.
Ras is a small GTP-binding protein in the cell that can be activated by many external stimuli, including epidermal growth factor and insulin (15
). The activated form of Ras, which binds to GTP, can associate with a number of downstream effectors to exert its biological effects. Among these effectors, the Raf-MEK-ERK pathway is the best characterized (31
). Raf is the mitogen-activated protein (MAP) kinase kinase kinase. After its activation by Ras, it will phosphorylate and activate MAP kinase kinases (MEKs), which will in turn phosphorylate MAP kinase or extracellular signal-regulated kinases (ERKs). Once activated, ERKs can translocate into the nucleus, where they will regulate the activities of transcription factors such as AP-1 (31
). The HBV X protein has been shown to activate this Ras-MEK-ERK signaling cascade (2
). Ras becomes inactivated when its associated GTP is converted to GDP.
As Ras is an important signaling molecule activated by many external stimuli, we investigated its possible effects on the replication of HBV. Our results demonstrate that activated Ras can suppress the replication of HBV through the MAP kinase pathway. This suppression by Ras apparently occurs at the transcription level and involves multiple regions of the HBV genome. It is also independent of the HBV X protein. These results provide a mechanism to explain how external factors may regulate HBV replication.