Interferon-inducible Dai Protein Inhibits Hepatitis B Virus Replication

Interferon-inducible DAI protein inhibits Hepatitis B virus replicationHepatitis B virus infection is a global public health problem, with over 300 million people worldwide being chronic HBV carriers and millions of deaths annually attributable to HBV related liver failure (cirrhosis or hepatocellular carcinoma). As a secreted cytokine that elicits distinct antiviral effect, interferon (IFN) has been used for treatment of chronic HBV infection for more than two decades, yet the mechanism of action of this antiviral remains poorly understood. Previous studies have proved that anti-virus action of interferon is mediated by the induction of anti-virus protein, plenty of genes are regulated by IFN, for example,2',5'-oligoadenylate synthetase, protein kinase R, and MxA and these products are the key mediators of IFN's antiviral response. Although previous studies have been demonstrated that a few of IFN-inducible proteins could exert anti-HBV effect in vitro or in vivo, such as APOBEC3, MxA and MyD88, they still could not fully account for IFN's effect on HBV replication. Hence, other IFN induced protein(s) might also be involved in the inhibition of HBV replication by IFN.DAI (DLM-1/ZBP1) was initially identified as a highly conserved RNA-binding protein, which could bind to the zipcode motif ofβ-actin mRNA and therefore be responsible for its intracellular localization and cell polarity. Francis V. Chisari et al have reported that the expression level of DAI's mRNA could be up-regulated after IFN-α/βand IFN-γstimulation in the highly differentiated hepatocyte HBVMet-4 harboring HBV replication. In 2007, Tadadsugu Taniguchi et al have demonstrated that DAI could significantly induce the expression of typeⅠIFN through binding to IRF3 and TBK1 as a DNA sensor. Therefore, it is quite possible that DAI might role in the inhibition of HBV replication exerted by typeⅠandⅡIFN.To verify DAI can induced by interferon in Human Hepatoma Cell Line(Huh7), we treat Huh7 cells with different dose of interferon-α. We found that IFN-a treatment can greatly activate DAI transcription.To determine the anti-HBV effect of DAI protein, we co-transfected Huh7 human hepatoma cells with an HBV-producing plasmid HBV 1.3 and either a control vector or vectors encoding HA tagged forms of DAI protein. Transfect ion rates were controlled by the inclusion of a GFP-expressing plasmid. To examine the effect of DAI on the synthesis of HBV proteins,48h after transfection, supernatants were collected and assayed for HBV surface antigen (HBsAg) and e antigen (HBeAg) by ELASA (Enzyme-Linked ImmunoSorbent Assay). Compare with the vector, the secretion of both HBsAg and HBeAg was significantly reduced in DAI transfect cell. To investigate whether the reduction of HBV protein synthesis was associated with the reduction of viral RNA transcripts, Northern blot analysis and real-time PCR was used to examine the cytoplasmic HBV RNAs in Huh7 cell after transfection with pHBV1.3 for 48h. The results show that DAI can reduce the HBV RNA in a dose-dependent manner. To investigate whether the HBV DNA replication was inhibited by DAI protein, viral replicative intermediates DNA extracted from core particles 48h after DAI transfection and then quantified by real-time PCR and Southern blot. We found that DAI can significantly reduce the HBV DNA replication.Poly(dA-dT).poly(dT-dA) (B-DNA) was identified as a DAI stimulator, I want to known is if B-DNA can induce endogenous DAI transcription and inhibit HBV replication? We add B-DNA to cell culture supernatant after HBV plasmid was transfect to Huh7 cell. Our result shows that B-DNA can induce DAI transcription and inhibit HBV replication in Huh7, although not as significantly as DAI over-expression.We future tudy the mechanism of DAI inhibit HBV repliacation.DAI have been implicated as a DNA sensor and interferon stimulator. So we determine whether the anti-HBV effect of DAI protein is interferon dependent. Taniguchi et al have shown that DAI can interact with TBK1 then recruit and activate IRF-3 result in producing interferon. So we want to know whether over-expression TBK1 can improve the anti-HBV effect of DAI. The results show that TBK1 can not affect the anti-HBV effect of DAI. IRF-3 was downstream of TBK1 and Taniguchi et al have shown that DAI (DLM-1/ZBP1) is critical for B-DNA-mediated IRF3 activation.So we speculate that IRF3 maybe play a role in DAI mediated anti-HBV effect. To test this idea, we block the IRF-3 with an IRF-3 domain negative plasmid. The result shows that the anti-HBV effect of DAI was not affected by this IRF-3 inhibitor.NF-κB is the key transcription factor in innate inmmune system. It is report that that DAI is necessary in B-DNA-mediated NF-κB binding activity.We first detects if NF-κB activity can be activated by DAI. As expected, DAI showed increased activation of NF-κB in dose dependent manner. Then we block the NF-κB activation with a NF-κB inhibitory plasmid IκBα-SR.When the activity of NF-κB was block, the anti-HBV effect of DAI is completely disappearing. So we speculate that the anti-HBV effect of DAI is related to activation of NF-κB.NF-κB activation can produce many kinds of cytokines inclucing interferon. To test whether anti-viral effect of DAI is associated with producing interferon or other cytokines, we first detect the interferon secret in supernatants by ELISA. The result shows that DAI can not triggers the secretion of interferon. To confirm that DAI inhibit HBV replication is not dependent on producing interferon or other cytokines, supernatant transfer experiment was performed in which cultured media were harvested from Huh7 cells transfected with plasmids expressing DAI and applied onto pHBV1.3 transfected Huh7 cells. It is show that the viral RNA levels were not affected in Huh7 cells treated with this conditioned medium. This result suggesting that inhibition of HBV replication by DAI is an intracellular event and independent of secreted cytokines.In summary, our studies demonstrated that the interferon inducible protein DAI can inhibit HBV replication. We also document that the DAI inhibit HBV replication is associated with the activation of NF-κB signaling pathway which may contributes to a better understanding of the mechanisms of interferon-induced inhibition of HBV replication and provide a new approach in the control of HBV infection.