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. 2012 Nov 6;109(45):18471-6.
doi: 10.1073/pnas.1204668109. Epub 2012 Oct 22.

Hepatitis B virus X protein targets Bcl-2 proteins to increase intracellular calcium, required for virus replication and cell death induction

Xin Geng  1 , Chenghao Huang, Yan Qin, Janet E McCombs, Quan Yuan, Brian L Harry, Amy E Palmer, Ning-Shao Xia, Ding Xue
Affiliations

Hepatitis B virus X protein targets Bcl-2 proteins to increase intracellular calcium, required for virus replication and cell death induction

Xin Geng et al. Proc Natl Acad Sci U S A. .

Abstract

Infection with the hepatitis B virus (HBV) promotes the development of hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) and is a leading cause of morbidity and mortality worldwide. HBV X protein (HBx) is an important effector for HBV pathogenesis, but its cellular targets and acting mechanisms remain elusive. We show here that HBx interacts with the anti-apoptotic proteins Bcl-2 and Bcl-xL through a Bcl-2 homology 3 (BH3)-like motif in mammalian cells. Importantly, mutations in the BH3-like motif that prevent HBx binding to Bcl-2 and Bcl-xL abrogate cytosolic calcium elevation and cell death induced by HBx expression in hepatocytes and severely impair HBV viral replication, which can be substantially rescued by restoring cytosolic calcium. These results suggest that HBx binding to Bcl-2 family members and subsequent elevation of cytosolic calcium are important for HBV viral replication. Consistently, RNAi knockdown of Bcl-2 or Bcl-xL results in reduced calcium elevation by HBx and decreased viral replication in hepatocytes. Our results suggest that HBx targets Bcl-2 proteins through its BH3-like motif to promote cytosolic calcium elevation, cell death, and viral replication during HBV pathogenesis, which presents an excellent therapeutic intervention point in treating patients with chronic HBV.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
HBx binds Bcl-2 and Bcl-xL and induces elevation of cytosolic Ca2+ in human hepatocytes through its BH3-like motif. (A) HBx associates with Bcl-2 and Bcl-xL in HepG2 cells. A coimmunoprecipitation (co-IP) experiment was performed in HepG2 cells transfected with pcDNA3.1-Flag-HBx or pcDNA3.1-Flag-HBx(G124L, I127A) (Materials and Methods). The cell lysate was precipitated with an anti-Flag antibody and analyzed by immunoblotting (IB), using anti-Flag, anti-Bcl-2, anti-Bcl-xL, and anti-Mcl-1 antibodies, respectively (lanes 3 and 4). One portion of the cell lysate was used in immunoblotting analysis to examine the expression levels of the HBx protein and Bcl-2 family proteins (lanes 1 and 2). (B) HBx expressed from its native promoter in a replicating HBV genome binds Bcl-2 and Bcl-xL in HepG2 cells. The co-IP experiment was performed as in A. The cell lysate was precipitated with an anti-HBx monoclonal antibody (16F9) and analyzed by IB analysis as in A. (C) Expression of HBx in HepG2 cells increases cytosolic Ca2+ through the BH3-like motif. HepG2 cells cotransfected with pcDNA3-mCherry and pcDNA3.1-Flag-HBx or pcDNA3.1-Flag-HBx(G124L, I127A) were treated with 4 μM Fura-2-AM 48 h posttransfection. Ca2+ concentrations were calculated from the measured Fura-2 ratios as previously described (27). Error bars indicate SEM (n > 12). **P < 0.01.
Fig. 2.
Fig. 2.
HBx induces cell killing in human hepatic cells through its BH3-like motif. HepG2 cells were transfected with 1 μg of empty pcDNA3.1 vector (A), pcDNA3.1-Flag-HBx (B), or pcDNA3.1-Flag-HBx(G124L, I127A) (C), using pEGFP-C1 (1 μg) as a cotransfection marker. Only the HBx-transfected cells (GFP positive) are shown (Materials and Methods). Lower Left square indicates the percentage of living cells, Lower Right square indicates the percentage of apoptotic cells, and Upper Right square indicates the percentage of necrotic cells.
Fig. 3.
Fig. 3.
Interaction between HBx and Bcl-2 proteins is critical for HBV DNA replication in human hepatic cells. (A) Southern blot analysis of HBV DNA replication in HepG2 cells transfected with the wild-type or mutant (G124L, I127A) pHBV replicon 3 d posttransfection. rc/ds DNA represents relaxed circular DNA and double-stranded DNA. ssDNA represents single-stranded DNA. (B) Measurement of the levels of the HBV core protein (HBcAg) in transfected HepG2 cells. The difference between cells transfected with the wild-type and mutant (G124L, I127A) pHBV is shown as fold change to wild type. The data represent mean ± SD from three independent experiments. ***P < 0.0001. (C) Northern blot analysis of HBV transcription in HepG2 cells transfected with the wild-type or mutant pHBV replicon 3 d posttransfection. Different HBV mRNAs are indicated. GFP mRNA from a cotransfection marker and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were used as controls. (D) Quantitative PCR analysis of HBV DNA replication in HepG2 cells transfected with the wild-type or mutant pHBV replicon with or without 5 μM ionomycin treatment. Three days posttransfection, cytoplasmic HBV viral particles were isolated and the viral DNA replication intermediates were quantified by real-time PCR (Materials and Methods). The results represent the fold change of the replicative intermediates from the mutant pHBV replicon compared with those from the wild-type pHBV replicon in HepG2 cells, using two different primer sets (one specific to the HBS ORF and one specific to the polymerase ORF). Data are presented as mean ± SEM. At least three independent experiments were performed for each dataset. ***P < 0.0001.
Fig. 4.
Fig. 4.
Interaction between HBx and Bcl-2 proteins is critical for HBV DNA replication in mouse hepatocytes. (A) Southern blot analysis of HBV DNA replication in mouse livers after hydrodynamical injection of the wild-type or the mutant (G124L, I127A) pHBV replicon 2 d postinjection. pcDNA3-GFP was coinjected as an injection marker. Equivalent amounts of liver tissues from six BALB/C mice in each injection group were collected and subjected to HBV DNA and HBcAg analyses (Materials and Methods). The levels of GFP and β-tubulin were determined by immunoblotting (IB) and used as controls to normalize the transfection efficiency. (B) Quantification of viral DNA intermediates from the Southern blot analysis in A. The results represent the relative value of different HBV DNA intermediates to the GFP control. The amounts of DNA and GFP were quantified by Quality One software (Bio-Rad). Data are presented as mean ± SEM. **P < 0.01. (C) A plot showing the levels of the HBV core protein (HBcAg) in mouse livers. All mice (n = 6) from each injection group were subjected to HBcAg analysis (Materials and Methods). RLU represents relative luminescence units (mean ± SEM). **P < 0.01.
Fig. 5.
Fig. 5.
Bcl-2 and Bcl-xL are important for HBV DNA replication. (A and B) (Right) HepG2 cells infected by lentivirus expressing control, Bcl-2, or Bcl-xL shRNA were transfected with the pHBV replicon and subjected to Q-PCR analysis as described in Fig. 3D. (Left) One portion of the cells was analyzed by immunoblotting to examine the expression levels of Bcl-2 and Bcl-xL, using α-tubulin as a loading control. Data are presented as mean ± SEM. ***P < 0.0001. (C) A working model of HBx-dependent viral pathogenesis. HBx directly interacts with Bcl-2 and Bcl-xL to increase cytosolic Ca2+. Increased cytosolic Ca2+ then promotes HBV replication and cell death. This signaling pathway is conserved in C. elegans.
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