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. 2017 Oct 6;8(56):96027-96034.
doi: 10.18632/oncotarget.21630. eCollection 2017 Nov 10.

Hepatitis B virus X protein inhibits apoptosis by modulating endoplasmic reticulum stress response

Jia Li  1   2 , Jiang He  3   4 , Yongming Fu  1 , Xingwang Hu  1 , Lun-Quan Sun  3   4 , Yan Huang  1 , Xuegong Fan  1
Affiliations

Hepatitis B virus X protein inhibits apoptosis by modulating endoplasmic reticulum stress response

Jia Li et al. Oncotarget. .

Abstract

Chronic Hepatitis B virus (HBV) infection is a major risk of hepatocellular carcinoma (HCC) worldwide. Hepatitis B virus X protein (HBx) is encoded by one of the four open reading frames of HBV, and is well known as an important coactivator for HBV replication and HBV-associated hepatocellular carcinogenesis. However, its role in keeping cells from apoptosis to promote HCC proliferation remains controversial. Here, we used HBx expressing HCC cells as a model, to investigate the mechanism of HBx-mediated cellular response to endoplasmic reticulum (ER) stress. We found that HBx protein was localized in ER lumen and interacted with GRP78 directly. This interaction resulted in suppression of eIF2α phosphorylation, inhibited expression of ATF4/CHOP/Bcl-2, and reduced cleavage of poly ADP-ribose polymerase (PARP) and level of γH2AX, thus preventing HCC cells from cell death and negatively regulating DNA repair. This study reveals a novel mechanism of the HBx-mediated oncogenesis and provides a basis for potential HBx-targeted therapeutic intervention of HCC.

Keywords: ER stress; HBV; HBx; apoptosis; hepatocellular carcinoma.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. HBx localization on ER
(A) HepG2 cells were co-transfected with pcDNA 3.1-GFP-HBX and pDsRed2-ER plasmids for 24h. Cells were rinsed and fixed. The coverslips were mounted and imaged using a laser scanning confocal microscope. (B) HepG2 cells were co-transfected with pcDNA 3.1-flag-HBX and pDsRed2-ER plasmids for 24 h. Cells were rinsed, fixed, permeabilized and blocked with BlockAid™ blocking solution. After labeling with anti-flag antibody overnight at 4°C, cells were washed in PBS and incubated with Alexa Fluor-conjugated secondary antibodies for 45 min at room temperature. The coverslips were mounted with DABCO anti-fade agent on glass slides and imaged using a laser scanning confocal microscope.
Figure 2
Figure 2. Direct interaction between HBx and Grp78
(A) Protein was extracted from the HepG2 cells expressing HBx or control cells and immune-precipitated with Anti-HBx antibody. The precipitates were subjected to immune-blotting by Anti-HBx and Anti-Grp78 antibodies. Loading control was indicated as Input. (B) Proximity ligation assay in HepG2 cells with or without HBx expression under Tg treatment up to 12 h. The red spots show sites of proximity ligation assay amplification reflecting the interaction between HBx and Grp78.
Figure 3
Figure 3. HBx impact on IRE1a and ATF6 pathways
HepG2 cells expressing HBx or control cells were treated for the indicated time with Tg (A) or Tm (B). The protein was extracted and subjected to Western blotting (D) and RNA was extracted for RT-PCR of ATF6 expression (C).
Figure 4
Figure 4. Effect of HBx expression on PERK branch under ER stress condition
(A) HepG2 cells expressing HBx or control cells were treated for the indicated time with Tg (A) or Tm (B), and p-eIF2a and ATF4 protein levels were measured by Western bots. Relative expression of ATF4 mRNA was detected by RT-PCR assay (C).
Figure 5
Figure 5. HBx inhibition of apoptosis and DNA repair
HepG2 cells expressing HBx or control cells were treated for the indicated time with Tg (A, C, E, F) or Tm (B, D). Expression of CHOP (A and B), cleaved PARP (C and D) and γ-H2AX and Bcl-2 were measure by Western blots. The Tg-treated HepG2 cells were assayed for apoptosis by TUNEL staining (F).
See this image and copyright information in PMC

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