Activation and inhibition of cellular calcium and tyrosine kinase signaling pathways identify targets of the HBx protein involved in hepatitis B virus replication

Abstract

Human hepatitis B virus (HBV) HBx protein is a multifunctional protein that activates cellular signaling pathways and is thought to be essential for viral infection. Woodchuck HBV mutants that lack HBx are unable to replicate in vivo or are severely impaired. HBV replication in HepG2 cells, a human hepatoblastoma cell line, is stimulated 5- to 10-fold by HBx protein. We have utilized the HepG2, HBx-dependent HBV replication system to study the effects of activators and inhibitors of cytosolic calcium and tyrosine kinase signaling pathways on viral replication. By transfecting either a wild-type HBV genome or an HBV genome that does not express HBx and then treating transfected cells with activators or inhibitors of signaling pathways, we identified compounds that either impair wild-type HBV replication or rescue HBx-deficient HBV replication. Geldanamycin or herbimycin A, tyrosine kinase inhibitors, blocked HBV replication. Derivatives of cyclosporine, i.e., cyclosporine A, cyclosporine H, and SDZ NIM811, which block cytosolic calcium signaling and specifically the mitochondrial permeability transition pore (SDZ NIM811), also impaired HBV replication. Treatment of cells with compounds that increase cytosolic calcium levels by a variety of mechanisms rescued replication of an HBx-deficient HBV mutant. Transcription of viral RNA and production of viral capsids were only minimally affected by these treatments. These results define a functional signaling circuit for HBV replication that includes calcium signaling and activation of cytosolic signaling pathways involving Src kinases, and they suggest that these pathways are stimulated by HBx acting on the mitochondrial transition pore.

FIG. 1.

HepG2 cell cytotoxicities of compounds used in this study. Cells were treated for 4 days with all compounds as described in Materials and Methods. The medium was replaced every 24 h with fresh agent. Cell death was quantified by LDH release into the medium, which was performed in duplicate on treated plates. Three independent experiments were conducted to calculate LDH release, percent cell death, and standard deviations. Cells lysed in 0.5% NP-40 represent the normalized release of maximal LDH. CsA, CsH, and SDZ NIM811 (NIM811) were used at 1 μg/ml, herbimycin A was used at 1 μM, 3TC was used at 2 μM, geldanamycin was used at 300 nM, glibenclamide was used at 15 μM, and gliotoxin was used at 0.5 μM. DMSO, dimethyl sulfoxide.

FIG. 2.

Glibenclamide and gliotoxin rescue replication of an HBV mutant which lacks HBx expression. (A and B) HepG2 cells were transfected with genomic DNA from an HBV HBx(−) mutant and treated with 15 μM glibenclamide (gilbencl.) (A) or 0.5 μM gliotoxin (B) for 4 days. Other plates of cells were transfected with wild-type HBV genomic DNA and similarly treated with glibenclamide. Southern blot DNA analysis of core particle-associated HBV DNA and Northern blot analysis of poly(A)⁺ RNA from cells treated with glibenclamide (A) or gliotoxin (B) are shown. Hybridization was performed with ³²P-labeled probes prepared from full-length HBV genomic DNA or β-actin to control for RNA levels. Blots were visualized by autoradiography and quantified by densitometry. (C) In vitro endogenous polymerase assay with cytoplasmic HBV core particles isolated from gliotoxin- or glibenclamide-treated cells. CytoplasmicHBV capsids were isolated from equal numbers of cells transfected with wild-type HBV or HBV HBx(−) genomic DNA and treated with agents as described above. Endogenous viral polymerase activity (upper panel) was measured by incorporation of α-³²P-labeled deoxynucleoside triphosphates in vitro. DNAs were resolved by agarose gel electrophoresis and detected by autoradiography. The lower panel shows the corresponding Western immunoblot analysis of isolated core particles resolved by native agarose gel electrophoresis and detected by anticore antibodies Representative results are shown.

FIG. 3.

Compiled results of effect of calcium agents and tyrosine kinase inhibitors on wild-type HBV and HBV HBx(−) DNA replication. The average enhancement or inhibition of HBV DNA replication that was observed after treatment with the indicated compounds is shown. The autoradiograms of from three or four independent experiments were quantified by densitometry and averaged, and standard deviations were calculated. Results were normalized to the untreated HBV-transfected control, which was set at 100% in all experiments. Calculated standard deviations are shown. (A) Replication activation relative to untreated HBV HBx(−)-transfected cells. (B) Replication inhibition relative to untreated wild-type HBV-transfected cells.

FIG. 4.

Effects of geldanamycin, herbimycin A, and 3TC on HBV DNA replication and mRNA levels. HepG2 cells were transfected with wild-type (wt) HBV genomic DNA and treated for 4 days with 300 nM geldanamycin, 1 μM herbimycin, or 2 μM 3TC. (A) Southern blot DNA analysis of core particle-associated HBV DNA and Northern blot analysis of poly(A)⁺ RNA were carried out as described in the legend to Fig. 2. Autoradiograms were quantified by densitometry. (B) An endogenous in vitro polymerase assay was performed on HBV particles isolated from equal numbers of cells, as described in the legend to Fig. 2. The upper panel shows the endogenous polymerase assay, and the lower panel shows Western immunoblot analysis of corresponding isolated core particles.

FIG. 5.

Effect of cyclosporines with different calcium-inhibitory mechanisms on wild-type (wt) HBV DNA replication. HepG2 cells were transfected with wild-type HBV genomic DNA for 4 days, with 1 μg of CsA, CsH, or SDZ NIM811 per ml. (A) Southern blot DNA analysis of core particle-associated HBV DNA and Northern blot analysis of poly(A)⁺ RNA. (B) In vitro endogenous polymerase assay of HBV particles isolated from equal numbers of cells, conducted as described in the legend to Fig. 2. The upper panel shows the endogenous polymerase assay, and the lower panel shows Western immunoblot analysis of corresponding core particles.