Abstract
Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to analyze the host response to HBV using an inducible HBV-producing cell line, HepAD38. Twenty-three proteins were identified as differentially expressed with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real time RT-PCR and Western blotting as well as in HBV-infected human liver biopsies by immunohistochemistry. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium. Conversely overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-β1 (IFN-β1). In this connection, the IFN-β1-mediated 2′,5′-oligoadenylate synthetase and RNase L signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p = 0.019) as compared with their counterpart pretreatment liver biopsies. In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via the IFN-β1-2′,5′-oligoadenylate synthetase-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection.
Highlights
Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma
We revealed the changes of the proteomic profile of hepatocytes upon HBV replication using an inducible HBV-producing system
glucose-regulated protein 78 (GRP78) was revealed as an intracellular anti-HBV factor using the gain- and loss-of-function studies, which suggested a new defense against HBV in hepatocytes
Summary
Plasmid Constructs and siRNA Synthesis—The pHBV (1.1-fold HBV genome, genotype C) was used to produce HBV virion as described previously [9]. The final concentration of siRNAs was 50 nmol/ liter, and the ratio of transfected plasmids was pAAV-GRP78 or pAAV/EF1␣:pHBV:pSV40-RL ϭ 5:1:0.1. To detect the expression level of GRP78, 2 ϫ 106 HepG2 cells were transfected with 5 g of pHBV or empty vector by nucleofection using Cell Line NucleofectorTM kit V and an Amaxa Nucleofector II device (program T-28, Amaxa Biosystems). For detecting the expression levels of IFNs and IFN-inducible genes triggered by GRP78 overexpression, HepG2 cells were nucleofected with pAAV-EGFP or pAAVGRP78 as described above. Cells were transferred to 6-well plates and left for incubation for 48 h before real time RT-PCR and Western blot analysis.
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