Molecular Mechanism Of The Inhibition Of Type Ⅰ Interferon Induction By Hepatitis B Virus Polymerase

Molecular Mechanism of TypeⅠInterferon Induction Inhibited by Hepatitis B Virus PolymeraseHepatitis B virus (HBV) is still one of the most dangerous pathogens jeopardizing public health globally. True, the routine vaccination has significantly decreased the risk of HBV infection in the population of newborn. But few and rare proper approaches can be clinically used to control HBV proliferation in those who have been chronically infected. More sadly,70% of patients with chronic HBV infection have no response to interferon a (IFNa) which is proved to effectively inhibit viral genes expression and proliferation. However, the molecular mechanism remains hazy. Thus, more investigation is needed to undertake to uncover the interplay between HBV and typeⅠIFN.TypeⅠIFN (including IFNa and IFNβ), playing a critical role in control and clearance of viral infection, can be induced by cellular pattern recognition receptors, such as Toll-like receptors (TLRs), RIG-I like helicases. Once engagement with viral ligands, TLR and RIG-I will recruit specific adaptors, for instance, TRIF, IPS1 and further motivate kinases TBK1/IKKεwhich can phosphorylate and activate interferon regulatory factor 3(IRF3), a key transcriptional factor required for typeⅠIFN production. In addition, DEAD-box helicase DDX3 is also another crucial component in the program of typeⅠIFN induction by TBK1/IKKε.In the long trajectory of evolution, viruses have developed numerous strategies to avert typeⅠIFN induction. For example, hepatitis C virus encodes NS3A to cleave key adaptors IPS1 and TRIF to sabotage typeⅠIFN synthesis. Similarly, a growing body of evidence also suggests that HBV can abrogate typeⅠIFN. HBV surface antigen (HBsAg), e antigen (HBeAg) and virions have been reported to almost completely suppress TLR-mediated antiviral activity and cytokine induction in murine liver parenchymal cells, indicating that HBV can counteract the TLR-mediated innate immune response. However, little is known about whether and how HBV disturbs cytoplasmic RIG-I-mediated IFNβinduction in human hepatocytes:In this study, we performed a functional screen assay to determine whether cytoplasmic viral proteins (including core, X and Pol) interfere with IFNP induction triggered by Sendai virus (SeV) and Newcastle Disease virus (NDV) challenge in primary human hepatocytes PH5CH8. Our results showed that only Pol inhibited the activation of IFNβpromoter in response to SeV and NDV infection though the expression of Core or X was higher than Pol. Interestingly, Pol also inhibited the activation of TLR3 signaling when poly(I:C) was directly added into cell culture, suggesting that the site of inhibition by Pol could be at the lower level in typeⅠIFN induction axis. Furthermore, we excluded the possibility of non-specific effects on IFNβpromoter using the unrelated p53 promoter reporter system. To further characterize the inhibitory effect of Pol, the induction of endogenous IFNβin PH5CH8 cells with or without Pol expression was examined using real-time quantitative PCR and ELISA. The data showed Pol could impede endogenous IFNβinduction in a dose-dependent manner. In addition, ectopic expression of Pol impaired the transferrable antiviral capacity of primary hepatocytes PH5CH8 in response to NDV infection which can protect Vero cells from NDV-GFP infection via secretion of IFNβ.To uncover the level at which Pol interferes with typeⅠIFN induction, 293T cells were co-tranfected with key molecules in typeⅠIFN induction axis and Pol. We found that Pol blocked the activation of IFNβpromoter triggered by all key molecules tested except IRF3-5D mutant. Moreover, we also determined the effect of Pol on the activation of endogenous IRF3 using native-PAGE, western blot and immunofluorescence. Our results showed that Pol could abrogate endogenous IRF3 activation by SeV. Unfortunately, we didn’t detect the direct interaction between Pol and IRF3, implying that the inhibitory effect of Pol on IRF3 activation is indirect. Thus, we defined the inhibition at the level of TBK1/IKKε.Given that DDX3 is involved in TBK1/IKKεmediated typeⅠIFN induction and also participates in inhibition of HBV reverse transcription via interaction with Pol, we speculated that DDX3 could be the target in the inhibition of typeⅠIFN by Pol. Immunoprecipitation assay revealed that less of TBK1/IKKεwas probed in the DDX3 immunoprecipitation complex in the cells with pol expression than in those without pol expression. However, over-expression of DDX3 restored the inhibition of SeV or TBK1 mediated induction of typeⅠIFN by Pol. It indicates that Pol can dampen the interaction between TBK1/IKKεand DDX3 through the competitive binding to DDX3.Taken together, these findings reveal a novel role of HBV polymerase in HBV counteraction of IFNβproduction in human hepatocytes...