Mechanism(S) Of The Inhibition Of Hbv Replication By Integrator Complex Subunit 10

Hepatitis B virus infection is a global problem of public health. About 3500 million people are infected with HBV in the world. Approximately,1 millions people die from hepatitis B and its complications ecah year. Persistence HBV infection, may cause liver cell damage and liver fibrosis, which develop into cirrhosis and hepatocellular carcinoma. The clear of viral infection or establishment of persistent infection depend on interaction between the virus and the host. Coincidently, previous studies have shown that host factors play critical roles in chronic HBV. Our previous studies of chronic HBV infection through genome-wide association study, which found that INTS10 located on human chromosome 8p21.3 region is a candidate gene with susceptibity to chronic HBV infection.To verify the hypothesis that INTS10 is a gene inhibiting HBV infection, we investigated the effect of INTS10 on HBV replication in hepatic cell lines. First, we co-expression pAAV-1.2HBV plasmid and INTS10 overexpression plasmid or control vector plasmid into the L-02 normal liver cell line and the HepG2 hepatoma cell line. Seven-two hours after transfection, the cells and supernatant were collected.The results suggest that INTS10 inhibited the accumulation of intracellular HBV replicative intermediates and expression levels of HBV RNA, and decreased the secretion of HBsAg or HBeAg. In addition to the L-02 and HepG2 cell lines, we did the same expriments in the HepG2.2.15 cell line with stable integration of HBV genome. We only expression INTS10 overexpression plasmid or control vector plasmid into the HepG2.2.15 cell line. Seven-two hours after transfection, the cells and supernatant were also collected to detect. The results suggest that INTS10 inhibits the accumulation of intracellular HBV replicative intermediates and expression levels of HBV RNA, but HBsAg or HBeAg have no significant change. Secondly we interfered the endogenous INTS10 expression in the L-02, HepG2 or HepG2.2.15 cell lines, and then transfected pAAV-1.2HBV plasmid into the L-02 or HepG2 cells, but not the HepG2.2.15 cells. In the L-02 or HepG2 cells, INTS10 downregulation can significantly enhance the replication levels of intracellular HBV replicative intermediates, upregulate expression levels of HBV RNA and the secretion of HBsAg or HBeAg. In the HepG2.2.15 cells, ENTS10 downregulation can also significantly enhance the replication levels of intracellular HBV replicative intermediates and expression levels of HBV RNA, but the secretion of HBsAg or HBeAg have no significant increase. All together, the results suggested that INTS10 play a important role of anti-HBV replication.INTS10 is a member of the integrator complex family. Integrator complex mediates the processing of small nuclear RNAs. Moreover, it is crucial for the splicing of pre-mRNA. In addition, it affects the persistent synthesis and expression of mRNA. However, it is still not been fully understood the role of Integrator complex in anti-HBV infection. To investigate the potential molecular mechanism of INTS10 in anti-HBV replication, we used the high-throughput gene expression profiling in non-tumor liver samples from 31 patients with HBV-related HCC. The significance analyses of microarrays (SAM) was performed to search for genes highly differential expressed between groups with high and low expression of INTS10, and DAVID tool was used for pathway enrichment analyses. The results show that the top two terms associated with the expression of INTS10 are spliceosome and retinoic acid-indueible gene-I-like receptor (RLR) signaling pathway (Pnominal= 1.5 × 10-5 and 1.8× 10-3, respectively).Although spliceosome is rarely reported to be involved in virus infection, the RLR members retinoic acid-induc ible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) senses HBV and activates innate immune signaling in hepatocytes to suppress virus replication. The NF-κB and IRF3 transcription factors activated by RLRs are essential components for transcriptional regulation of type I IFNs (such as IFN-a or IFN-β) and type Ⅲ IFNs (IFN-λ,, also known as IL-28 and IL-29), the central antiviral cytokines, and several other antiviral mediators and inflammatory effector genes. Therefore, we focus on the RLR pathway in this study and hypothesize that INTS10 might inhibit HBV infection via the NF-κB and/or IRF3 signaling.To validate the hypothesis, firstly, we co-transfected the L-02 cell or HepG2 hepatoma cell line with an pAAV-1.2HBV plasmid and PLV-INTS10-EGFP pasmid, while simultaneously down-regulated the INTS10 by siRNAs in cell lines, and then transfected the pAAV-1.2HBV plasmid. On the other hand, we transfected HepG2.2.15 with an PLV-INTS10-EGFP pasmid, or down-regulate the INTS10 by siRNAs.After seventy-two hours, the cells were collected for Western blot assay, in order to exam the effect of INTS 10 on RLR pathway. The result showed that the level of phosphorylated IRF3 (p-IRF3) was positively regulated by INTS 10 during HBV infection, while the level of p65/p-p65 or IκBa/p-IκBα was not changed with different levels of INTS10. Conversely, down-regulation of INTS10 by siRNAs led to significantly decreased activity of p-IRF3, without influencing activity of the p65/p-p65 or IκBα/p-IκBα.To further validate the results, we examined the effect of INTS10 on activity of the IFN-stimulated response element (ISRE) in reporter assays. we found that overexpression of INTS10 could potently activate ISRE reporter in HepG2.2.15 cells, L-02 cells or HepG2 cells. Conversely, down-regulation of INTS10 by siRNAs led to significantly decreased activity of ISRE reporter. Furthermore, we found that overexpression of INTS10 could elevate mRNA level of IFN-λ. Conversely, down-regulation of INTS10 by siRNAs led to significantly decreased mRNA level of IFN-X,.In the light of western blot assays and reporter assays, we validated that INTS10 can regulate activity of p-IRF3 and ISRE, then increase the mRNA expression of endogenous IFN-λ, thus inhibit the replication of HBV. To further confirm the fact that INTS10 can control HBV by up-regulate IFN-X in IRF3 pathway, we down-regulated IRF3 by siRNAs, and then overexpressed INTS10 to detect the change of HBV. Firstly, HepG2.2.15 cells was transfected with IRF3-specific siRNA, and 36 hours later, trans feted with INTS10-expressing plasmids.72 hours later, we found that the inhibition of INTS10 on levels of HBV DNA, HBV RNAs, and HBsAg and HBeAg was weakened. Taken together, these in vitro data suggest that INTS10 inhibits HBV infection partially in an IRF3-dependent manner.To further validate a role for INTS10 in facilitating human HBV clearance, we studied plasma samples from patients infected with HBV to identify an association between plasma INTS10 levels and an effective immune response in humans clearing HBV. We found significantly decreased levels of plasma INTS10 were observed in persistent HBV infected subjects (PIs) as compared with spontaneously recovered subjects (SRs), and expression levels of plasma INTS10 were negetive correlate with HBsAg. In addition, we conducted immunohistochemical detection in liver tissues from 14 patients with HBV self-limiting recovery and 40 patients with persistent HBV infection. Results showed INTS10 levels were positively correlated with p-IRF3 levels while INTS10 levels has no significant correlation with p-p65 levels. The results further demonstrating that INTS10 may be involved in HBV clearance via IRF3.In conclusion, our studies suggest that INTS10 up-regulates p-IRF3, then actives IFN-X, subsequently inhibits HBV replication. This study further clarified the role of IRF3 in RIG-I antiviral pathway, contributing to explain the mechanism by which INTS10 inhibits HBV replication.