Identification of a BH3-like domain in the core protein. (A) Alignment of the BH3 domain of the genotype 1b core protein with the BH3 domains of members of the Bcl-2 family. Numbers in parentheses represent the positions of amino acid residues of the respective proteins. The four hydrophobic amino acids that make critical contacts with residues in the BH3 recognition grooves present on the surfaces of the prosurvival Bcl-2 family proteins are indicated as h1 to h4. (B) Alignment of the core protein (residues 109 to 132) of different genotypes. The consensus sequences for these genotypes were obtained from http://hcv.lanl.gov/content/hcv-index. The highly conserved L and D residues, at positions 119 and 124 of the genotype 1b core protein, respectively, in BH3 domains are boxed.

Interaction of the core protein with prosurvival members of the Bcl-2 family determined by coimmunoprecipitation experiments. (A) Huh7 cells were transfected with cDNA constructs for expressing flag-GST (negative control), flag-core, or flag-coreΔ115-128aa, and myc-tagged prosurvival members of the Bcl-2 family (myc-Bcl-X_L [lanes 1 to 3], myc-Bcl-w [lanes 4 to 6], and myc-Mcl-1 [lanes 7 to 9]). The cells were harvested at ∼16 h posttransfection, lysed, and subjected to IP with anti-flag monoclonal antibody conjugated to Sepharose beads. The amount of myc-tagged proteins that coimmunoprecipitated (IP) with the flag-tagged proteins was determined by Western blot analysis (WB) with an anti-myc rabbit polyclonal antibody (top). The amounts of myc-tagged and flag-tagged proteins in the lysates before IP were determined by subjecting aliquots of the lysates to Western blot analysis (middle). The protein marked with an asterisk represents the heavy chain of the antibody used for IP (top), and the amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin (bottom). (B) Huh7 cells were transfected with cDNA constructs for expressing flag-core or flag-coreΔ115-128aa. IP then was performed using anti-Mcl-1 or anti-HA rabbit polyclonal antibodies and protein A agarose beads. The amounts of flag-tagged core protein in the lysates before IP (lanes 1 and 2) or coimmunoprecipitated (lanes 3 to 6) were determined by Western blot analysis with an anti-flag monoclonal antibody (top). Similarly, the amounts of endogenous Mcl-1 in these samples were detected using an anti-Mcl-1 monoclonal antibody (middle). The protein marked with an asterisk represents the heavy chain of the antibody used for IP (top), and the amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin (bottom).

Release of cytochrome c from isolated mitochondria by a combination of the core-BH3 and Bad-BH3 peptides or Noxa-BH3 and Bad-BH3 peptides. (A) Mitochondria isolated from 293T cells were incubated with either Bad peptide, core protein peptide, or a combination of the two peptides in equimolar concentrations. The total amount of peptide used in each experiment is indicated. Following centrifugation, the supernatants and pellets were subjected to Western blot analysis with anti-cytochrome c (cyt c) or anti-Hsp-60 antibodies. (B) The same experiment was repeated with Noxa peptide or a combination of Noxa and Bad peptides in equimolar concentrations.

Effects of alanine substitutions on the binding of the core protein to Mcl-1. (A) Huh7 cells were transfected with cDNA constructs (1.0 μg) for expressing flag-GST (negative control), flag-tagged wild-type core protein, or single-alanine-substitution mutants (L119A, V122A, and L126A). All cells were cotransfected with myc-tagged Mcl-1 (1.5 μg). (B) Huh7 cells were transfected with cDNA constructs for expressing flag-GST (negative control, 0.5 μg), flag-tagged wild-type core protein (0.5 or 1.0 μg), or single-alanine-substitution mutant D124A (0.5 or 1.0 μg). All cells were cotransfected with myc-tagged Mcl-1 (1.5 μg). Coimmunoprecipitation then was performed as described in the legend to Fig. 3A. The amount of myc-tagged proteins that coimmunoprecipitated (IP) with the flag-tagged proteins was determined by Western blot analysis (WB) with an anti-myc rabbit polyclonal antibody (top). The amounts of myc-tagged and flag-tagged proteins in the lysates before IP were determined by subjecting aliquots of the lysates to Western blot analysis (middle). The amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin (bottom). The protein marked with an asterisk represents the heavy chain of the antibody used for IP (top). Similar results were obtained in four independent experiments, and a representative set of data is presented.

Comparison of parental J6/JFH-1 and mutant J6/JFH-1(V119L) recombinant viruses. Huh7.5 cells were infected with recombinant HCV at a multiplicity of infection of 0.1 CIU/cell or with a mock preparation, and various assays were performed at different days after infection. (A) Cell viabilities were determined. (B) Caspase-3 activity per cell was determined. (C) The amount of DNA fragmentation per cell was determined. (D) The production of cell-free infectious virus particles was determined. (E) Virus spread in the culture was quantitated. (F) HCV RNA replication was determined by quantitative real-time PCR analysis. (G) Interaction of the core protein with Mcl-1 was determined by coimmunoprecipitation experiments at 3 days p.i. IP was performed using anti-Mcl-1 or anti-HA rabbit polyclonal antibodies and protein A agarose beads. The amounts of the core protein in the lysates before (lanes 1 and 2) and after IP (lanes 3 to 6) were determined by Western blot analysis with an anti-core monoclonal antibody (top). Similarly, the amounts of endogenous Mcl-1 in the samples were determined using an anti-Mcl-1 monoclonal antibody (bottom). Statistical analysis was performed using the one-way analysis of variance to determine if the differences between parental and mutant viruses were statistically significant, and those with P values of <0.05 (marked by asterisks) are considered statistically significant. Data were obtained from three independent experiments, each with triplicate cultures.

Induction of apoptosis by the overexpression of the core protein in Huh7 cells. (A) A CaspACE fluorometric assay system from Promega Corporation (Madison, WI) was used to measure the activation of caspase-3, which is a hallmark of apoptosis, in Huh7 cells that were transfected with vector only, a classical apoptosis inducer (Bax), the wild-type core protein, and a core protein mutant lacking the putative BH3 domain (coreΔ115-128aa). All experiments were performed in triplicate, and the average values with standard deviations are plotted. (B) Western blot analysis also was performed to determine the cleavage of endogenous PARP, which is a substrate of activated caspase-3, from 116 to 83 kDa (top). Similarly, the expression levels of the different proteins were determined using anti-flag antibody (middle). The amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin (bottom).

Effects of Mcl-1 and Bcl-X_L overexpression on the proapoptotic property of the core protein. (A) A CaspACE fluorometric assay system from Promega Corporation (Madison, WI) was used to measure the activation of caspase-3 in Huh7 cells that were transfected with Bcl-2, Bcl-X_L, Bcl-w, Mcl-1, or vector only. All experiments were performed in triplicate, and the average values with standard deviations are plotted. (B) Western blot analysis also was performed to determine the cleavage of endogenous PARP (top) and expression levels of the myc-tagged prosurvival members of the Bcl-2 family (middle). The amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin (bottom). (C) A CaspACE fluorometric assay system from Promega Corporation (Madison, WI) was used to measure the activation of caspase-3 in Huh7 cells that were singly transfected with vector, the wild-type core protein, Mcl-1, or Bcl-X_L, or that were cotransfected with wild-type core protein and Mcl-1 or Bcl-X_L. The amounts of flag-core and myc-Mcl-1 or myc-Bcl-X_L DNAs used in each of the transfections are indicated in micrograms. In each transfection, the total amount of DNA was normalized to 3 μg with the addition of empty vector if necessary. All experiments were performed in triplicate, and the average values with standard deviations are plotted. (D) Western blot analysis also was performed to determine the cleavage of endogenous PARP (top) and expression levels of myc-tagged Mcl-1 and Bcl-X_L and flag-tagged core protein (middle). The amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin (bottom).

Effects of alanine substitutions on the proapoptotic property of the core protein. (A) A CaspACE fluorometric assay system from Promega Corporation (Madison, WI) was used to measure the activation of caspase-3 in Huh7 cells that were transfected with vector only, wild-type core, or alanine substitution mutants. All experiments were performed in triplicate, and the average values with standard deviations are plotted. (B) Western blot analysis also was performed to determine the cleavage of endogenous PARP (top) and expression levels of the core proteins (middle). The amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin (bottom). (C) The levels of activated caspase-3 induced by the wild-type core protein and the D124A mutant in six independent experiments were compared using the two-tailed Student's t test, and the difference was found to be statistically significant (P < 0.05). The values from each of the experiments are plotted as open triangles, and the average values are plotted as solid lines.

Comparison of the proapoptotic properties of the core proteins of genotypes 1b and 2a. (A) A CaspACE fluorometric assay system from Promega Corporation (Madison, WI) was used to measure the activation of caspase-3 in Huh7 cells that were transfected with vector only, wild-type core of genotype 1b or 2a, or their substitution mutants. All experiments were performed in triplicate, and the average values with standard deviations are plotted. (B) Western blot analysis also was performed to determine the cleavage of endogenous PARP (top) and expression levels of the core proteins (middle). The amounts of total cell lysates loaded were verified by measuring the levels of endogenous actin (bottom).