est is how TGFb1 manipulates the binding of HNF-4a to the HBV core promoter. To confirm whether TGF-b1 purchase Foretinib 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 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
