est is how TGFb1 manipulates the binding of HNF-4a to the HBV core promoter. To confirm whether TGF-b1 treatment interferes with the interaction between HNF-4a and its responsive element, the binding activity of endogenous HNF-4a in the presence or absence of TGF-b1 was examined by EMSA analysis. An EMSA probe corresponding to the 59-HNF4BE located within HBV core 5 The Suppression of HBV Replication by TGF-b1 promoter was chose to perform EMSA analysis. The binding activity of endogenous HNF-4a to its responsive probe was significantly reduced after TGF-b1 treatment. We next investigated whether TGF-b1 affects the expression of HNF-4a in 1.3ES2 cells. Surprisingly, we found that TGF-b1 treatment resulted in a dramatic reduction in HNF-4a mRNA. Furthermore, a significant disappearance of HNF-4a protein by TGF-b1 treatment was observed by Western blot analysis. HNF-4a plays a crucial role in the suppressive effect of TGF-b1 on HBV replication To confirm the significance of HNF-4a protein in mediating the antiviral effect of TGF-b1, the expression level 
of endogenous HNF-4a was specifically reduced by RNA interference technique, and then the inhibitory effects of TGF-b1 were analyzed. Consistent with the previous study, the reduction of HNF-4a expression by RNAi was sufficient to trigger HBc The Suppression of HBV Replication by TGF-b1 repression even without TGF-b1 treatment. Although TGF-b1 significantly repressed HBc expression in these mock control cells, the level of TGF-b1-mediated HBc reduction in these HNF-4a knock-down cells was relatively less significant. Since the metabolism of HNF-4a by TGF-b1 treatment has been reported to be mediated through the proteasome-dependent degradation pathway, we next examined whether blocking the TGF-b1-mediated HNF-4a degradation by proteasome inhibitor MG132 could alter the consequence of HBc suppression by TGF-b1 treatment. We found that MG132 treatment not only protected HNF-4a protein from degradation but also prevented HBc from TGF-b1-mediated repression. Interestingly, Northern blot analysis revealed that the reduction of the 3.5 kb transcripts by TGF-b1 was also prohibited while cells were pretreated with MG132. Taken together, our data indicate that the manipulation of HNF-4a expression plays a crucial role in diminishing of HBc expression as well as pgRNA transcription during TGF-b1 treatment. 7 The Suppression of HBV Replication by TGF-b1 Recovery of HNF-4a expression in TGF-b1-treated cells can rescue HBV replication To further confirm that whether the expression level of HNF-4a is the key parameter in modulating of the HBV replication by TGF-b1, HBV-producing cells were induced to express different amounts of rat HNF-4a in the presence of TGF-b1. Our data revealed that ectopic expression of rat HNF-4a alone was sufficient to rescue the impaired PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22182644 HBc expression caused by TGF-b1 treatment, and that the assembling of intracellular viral particles could also be restored by rat HNF-4a overexpression. Moreover, the expression level of HBV transcripts and intracellular viral replicative intermediates were both elevated in parallel with the increased amounts of rat HNF4. In conclusion, we provide LY-2940680 supplier evidence to support our scenario in which the crucial hepatic transcription factor HNF-4a is indispensable for the suppressive effect of TGF-b1 on HBV replication. Discussion TGF-b1 inhibits HBV replication through the modulation of HNF-4a Our results indicate that TGF-b1 exerts its anti-HBV effect t