The Mechanism Of Hbs Protein Induces Negative Regulator HPIAS1 Of Interferon Signaling Pathways

PIAS1 protein, also known as Gu RNA helicase II binding protein (GBP), was belonged to the mammalian PIAS protein superfamily, originally characterized based on their interaction with the STAT family of transcription factors. Subsequent studies revealed that PIAS1 contains a conserved RING domain of the superfamily that has been linked to a function as a small ubiquitin-related modifier (SUMO) E3 ligase, modulates cellular processes such as cell proliferation, cell differentiation, DNA damage responses, and inflammation responses. While the SAP domain and the C-terminal region of PIAS1 function both at the virus-induced early signaling stage and IFN stimulated amplifying stage with distinct mechanisms. Deletion of PIAS1 leads to the enhancement of interferon-inducible genes and increased protection against infection. Recent studies have shown that increased hPIAS1 was developed in the nucleus of most hepatocytes in hepatitis C virus (HCV) tissue biopsy sections of the interferon treatment-nonresponder chronic HCV patients compared with the treatment-responder patients or healthy controls. Although HCV and Hepatitis B virus (HBV) are irrelevant, evidences of analogous molecular mechanism have been found on signal pathway mediators.HBV is a classical hepadnavirus and persistant viral infection can progress to serious complications, such as acute sever hepatitis, liver cirrhosis, and hepatocellular carcinoma. Approved treatments for chronic hepatitis B (CHB) include a few nucleos(t)ide analogues or alpha interferon (IFN-a), which converts CHB into inactive HBV infection in only 20-30% of the treated patients. The underlying molecular mechanisms responsible for the ineffectiveness of treatment remain unclear. Many reports have demonstrated the role of levels of HBV DNA in response to interferon therapy. High viral load seems associated with a low T-cell activation and poor response to interferon treatment in CHB patients. Intriguingly, further study found that the molecular basis of the IFN antagonist is somewhat because the hepatitis virus proteins impair the IFN-alpha-induced signal transduction by inducing series of negative mediators. Based on the preliminary clinical trials of our laboratory, we observed an entirely different hPIAS1 protein expression in both PBMCs and liver samples in CHB patients responded with the PegIFNa-2a treatment and in those of non-responders. Hence, we postulate that the hPIAS1 gene transcription is regulated by HBV proteins, one of the most frequent potential causes of the failure of interferon therapy.In this study, the hPIAS1 promoter/luciferase reporter plasmid hpiaslp(-1300)LUC was cloned and cotransfected with eukaryotic expression plasmids of HBV protein (pcDNA-HBs, pcDNA-HBx and pcDNA-HBc) or pcDNA3.1 empty vector separately. Cotransfection of pcDNA-HBs with hpiaslp(-1300)LUC respectively resulted in a significant increase of 2.9- and 3.6-fold relative luciferase activity in CHO and HepG2 cells, when compared with pcDNA3.1 empty vector cotransfected cells. We demonstrate that it's the HBs protein, but not the HBx or HBc protein, enhanced hPIAS1 transcription. Then, series of 5'-Truncation of hPIAS1 promoters were carried out using hpias1p(-1300)LUC as template. By promoter luciferase reporter assays, a strong regulatory region from -712 to-507 (relative to the transcriptional starting site) was responsible for hPIAS1 gene transcription in response to HBs. Binding of the TAL1, E47, Myogenin (MYOG) and NFI to cis-regulatory elements in the hPIAS1 promoter were confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Moreover, the TAL1, E47, MYOG and NFI were shown highly expressed and to be translocated to the nucleus in association with hPIAS1 expression in HBs-transfected HepG2 cells by immunofluorescence laser confocal microscopy assay. SiRNA interference of TAL1, E47, MYOG and NFI expression inhibited hPIAS1 gene transcription to 64.3%,58.0%,59.9% and 63.1% in response to HBs, respectively. Further study revealed that increased phosphorylation of ERK and p38MAPK was developed in HBs-transfected HepG2 cells, and ERK inhibitor PD098059 or p38MAPK inhibitor SB203580 abolished their nuclear TAL1, E47, MYOG and NFI expressions and hPIAS1 induction, whereas JNK inhibitor SP600125 had no such effect.Studies from patients with HBV infection have shown that phosphorylation of p38MAPK and ERK was markedly increased in PBMCs of the CHB patients and HBsAg carriers with high HBV levels (no matter the ALT and AST normal or not), in comparison with that in HBsAg carriers with HBV DNA undectable or healthy controls. What's more, though there was basal expression of the transcription factors TAL1, E47, NFI and MYOG in HBsAg carriers with HBV DNA undectable or healthy controls, they were highly expressed in PBMCs of HBsAg carriers with high HBV levels. Intriguingly, in liver tissue of CHB patients with high levels of HBV, a significant increase of hPIAS1 expression has also been observed, compared with those in CHB patients with low HBV levels or healthy controls.In summary, this study has demonstrated that the HBs initiates the transcription of hPIAS1 through the coactivators of TAL1, E47, NFI and MYOG, dependent on the activation of p38MAPK and ERK signal pathways, respectively. This work provides new insights into the interaction between HBV virus and the host gene hPIAS 1 expression, and potential anti-viral therapeutic targets for controlling diseases to which HBV contributed.