| Summary: | 博士 === 國立陽明大學 === 微生物及免疫學研究所 === 93 === In the first part of this thesis, we have evaluated the transcriptional efficiency of covalently closed circular DNA by using a RT-PCR combined with restriction enzyme digestion method. Virus persistence in chronic hepatitis B patient is manifested by sustaining a pool of covalently closed circular DNA (cccDNA) in the nucleus of the infected hepatocytes that presumably serves as a template for HBV genes expression. We used a modified 1.3-fold HBV genome, with a BclI-genetic marker embedded in the 5’-end redundancy region, to examine the transcriptional activity of cccDNA and the regulatory effect of HBx protein on the transcriptional activity. After harvesting total RNA from transfected cells or stable lines, we specifically monitored the transcription ability of cccDNA by using a RT-PCR combined with restriction enzyme digestion method. With this approach, we have found that (1) RT-PCR combined with digestion by BclI marker is highly specific and can distinguish transcripts originating from cccDNA from those originating from an integrated viral genome; (2) the transcriptional ability of cccDNA was shown to be less efficient than that from integrated viral genome; (3) the transcriptional activity of the cccDNA was significantly regulated by the HBx protein, a potential transcription activator. Accordingly, this information may provide a meam to further examine the transcriptional regulation of cccDNA and possibly, a very useful tool in the development of a cure for patients with a chronic HBV infection.
In the second part of this thesis, we focused on the elucidation of the anti-viral effect of Transforming Growth Factor-beta1 on HBV replication. The replication of hepatitis B virus (HBV) has been reported to be suppressed by several cytokines, such as alpha/beta interferon (IFN-□/□), gamma interferon (IFN-□), and tumor necrosis factor alpha (TNF-□). Here we described that transforming growth factor beta 1 (TGF-□1), one of the cytokines elevated in hepatitis, has potential antiviral activities to decrease HBV replication in vitro. Using a virion-producing cell line derived from HepG2, we showed that TGF-□1 could suppress HBV replication as demonstrated by dramatically reduction of extracelluar and intracellular nucleocapsid. Concurrently, the cytosolic viral replicative intermediates were significantly diminished after TGF-□1 treatment. Furthermore, we also have evidence suggests that TGF-□1 can suppress HBV particle assembly by interfering with pgRNA transcription and HBc translation. RNase protection analysis revealed that TGF-□1 inhibited viral replication primarily by downregulating the expression of pregenomic RNA (pgRNA), as well as hepatocyte nuclear factor(s). Following the attenuation of viral transcripts by TGF-□1, we found that the HBc protein synthetic rate was also reduced due to the failure of ribosome engagement. Taking together, our data demonstrated that the antiviral effect of TGF-□1 was primarily due to both the transcriptional and translational controls, and suggest that elevated TGF-□1 may play a critical role in suppressing viral replication in chronic hepatitis.
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