Effect of SPCS1 knockdown on the processing of HCV structural proteins and secretion of host proteins.

<p>(A) Core-NS2 polyprotein was expressed in KD#31 cells or parental Huh-7 cells. Core, NS2, SPCS1, and actin were detected by immunoblotting 2 days post-transfection. (B) Expression constructs of NS2 and NS2/3 proteins. His to Ala substitution mutation at aa 956 in NS2 is indicated by an asterisk. Gray circles and bold lines indicate FLAG-tag and the spacer sequences, respectively. Positions of the aa residues are indicated above the boxes. (C) Effect of SPCS1 knockdown on processing at the NS2/3 junction. Huh-7 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 30 nM, and then transfected with plasmids for FLAG-NS2, F-NS2-3, or F-NS2-3 with a protease-inactive mutation (H956A). NS2 in cell lysates was detected by anti-FLAG antibody 2 days post-transfection. Arrowhead indicates unprocessed NS2-3 polyproteins. (D) Effect of SPCS1 knockdown on the secretion of apoE. Huh7.5.1 cells were transfected with SPCS1 siRNAs or control siRNA at a final concentration of 20 nM, and apoE in the supernatant and SPCS1 and actin in the cells were detected 3 days post-transfection. (E) Effect of SPCS1 knockdown on the secretion of albumin. Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA, and albumin in the culture supernatants at 2 and 3 days post-transfection was measured by ELISA.</p

ATP consumption in cells replicating HCV RNA.

<p>(Left) The indicated cell lines were pretreated with 10 µM PSI-6130 for 3 days or were cultured in the absence of the drug, followed by trypsinization and permeabilization. ATP-containing reaction buffer plus 10 µM PSI-6130 was added to some of the non-pre-treated cells (PSI-6130, 15 min; light gray bars). ATP-containing PSI-6130-free reaction buffer was added to the rest of the non pre-treated cells (PSI-6130, (−); white bars) and to the pre-treated cells (PSI-6130, 3 days; dark gray bars). After 15 min incubation, ATP levels in cell lysates were measured using a luciferase-based assay. ATP reduction compared to ATP levels at the 0-time point was calculated. The mean values of three independent samples with SD are displayed. Statistical differences between cells treated with and without treatment with PSI-6130 were evaluated using Student's <i>t</i>-test. (Right) HCV RNA titers in cells corresponding to the left panel were determined using real-time quantitative RT-PCR. Data are presented as means and SD for three independent samples. NTD indicates not detected.</p

Estimation of ATP levels at possible sites of HCV RNA replication in living cells.

<p>(A) Venus/CFP emission ratios were calculated from images of CFP and Venus channels in individual cells for each group. Bar- and dotted graphs indicate ratios within the cytoplasm and ratios for dot-like structures, respectively, in the same cells, as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002561#ppat-1002561-g005" target="_blank">Figures 5A and 5B</a>. Data in bar graphs are indicated as means and SD. Horizontal lines in the dot graphs denote means from at least three independent cells. Values in the cytoplasm of cells transfected with NS5A-AT1.03^YEMK and SGR-AT1.03^YEMK were statistically significant (p<0.05) as evaluated using the Student's <i>t</i>-test. (B) Calibration of NS5A-ATeam in cells under semi-intact conditions. Cells were transfected with NS5A-AT1.03 and NS5A-AT1.03^YEMK, respectively. Forty-eight hours later, the cells were permeabilized, followed by addition of known concentrations of ATP. FRET analyses were performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002561#ppat-1002561-g005" target="_blank">Figure 5A</a>. Each trace represents mean with SD of at least six independent cells. Plots were fitted with Hill equations with a fixed Hill coefficient of 2; R = (R_max−R_min)×[ATP]²/([ATP]²+<i>Kd</i>²)+R_min, where R_max and R_min are the maximum and minimum fluorescence ratios, respectively. <i>Kd</i> is the apparent dissociation constant. R values were 0.994 and 0.986 for NS5A-AT1.03 and NS5A-AT1.03^YEMK, respectively. (C) Cells were transfected with NS5A-AT1.03, SGR-AT1.03^RK or SGR-AT1.03. The cells were then treated with PSI-6130 at indicated concentrations (µM) for 10 min or 2 h, and were analyzed as described in (A). Values in the cytoplasm of cells transfected with SGR-AT1.03 with and without PSI-6130 treatment were statistically significant (p<0.05 for control versus 0.1 or 1 µM PSI-6130, p<0.01 for control versus 0.5 or 5 µM PSI-6130) as evaluated using the Student's <i>t</i>-test. Representative cells treated with 5 µM PSI-6130 are shown in the right panel. The lower panel is a five-fold magnification of the boxed area. Scale bars, 20 µm.</p

Development of NS5A-ATeam and SGR-ATeam to enable real-time monitoring of ATP.

<p>(A) Schematic representation of the ATeam and NS5A-ATeam used in this study. ATeam genes were inserted into the 3′ region of a HA-NS5A expression vector to generate NS5A-ATeam. The underlined sequences indicate NS5A residues. The insertion site was between residues 2394 and 2395, numbered according to the polyprotein of the HCV JFH-1 isolate. CMV, Cytomegalovirus promoter; CAG, CAG promoter; ATP b.p, ATP binding protein. HA, HA tag. (B) Huh-7 cells were transfected with ATeam and NS5A-ATeam constructs. Forty-eight hours post-transfection, the Venus/CFP ratios of each cell were calculated from fluorescent images acquired with a confocal microscope in the same way as described in the legends for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002561#ppat-1002561-g002" target="_blank">Figure 2</a>. Each plot shows the ratio of individual cells. Horizontal lines represent means. (C) Schematic representation of the SGR and SGR-ATeam plasmids used, with or without the firefly luciferase gene (Fluc). HCV polyproteins are indicated by the open boxes. ATeam genes were inserted into the same site in the NS5A C-terminal region. Bold lines indicate the HCV UTR. EMCV IRES is denoted by the gray bars. Pol I P, Pol I promotor; dC, 5′ region of Core gene; Pol I T, Pol I terminator. (D) Replication levels of SGR/luc-AT1.03 in transfected cells were determined by luciferase assay 1–5 days post-transfection. SGR/luc and SGR/luc-GND were used as positive and negative controls, respectively. Values given were normalized for transfection efficiency with luciferase activity determined 24 h post-transfection. All data are presented as means and SD for three independent samples. (E) Huh-7 cells were transfected with constructs encoding NS5A, NS5A-AT1.03, SGR, SGR-AT1.03, SGR/luc or SGR/luc-AT1.03, followed by immunoblotting with anti-NS5B or anti-beta-actin antibody. (F) Cells transfected with constructs encoding NS5A, NS5A-AT1.03, SGR or SGR-AT1.03 were analyzed by immunoblotting with anti-NS5A, anti-NS5B or anti-beta-actin antibodies.</p

Interaction of HCV NS2 protein with SPCS1 in mammalian cells.

<p>(A) Expression constructs of SPCS1-myc and FLAG-NS2 used in this study. TM regions are represented as gray. Myc-tag regions are depicted by the black circles. Gray circles and bold lines indicated FLAG-tag and spacer (GGGGS) sequences, respectively. Adaptive mutations are indicated as asterisks. Positions of the aa resides are indicated above the boxes. (B) 293T cells were co-transfected with a FLAG-tagged NS2 expression plasmid in the presence of a SPCS1-myc expression plasmid. Cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in immunoprecipitation (IP) were examined by immunoblotting using anti-FLAG- or anti-myc antibody. An empty plasmid was used as a negative control. (C) HCVcc infected Huh-7 cells were transfected with a SPCS1-myc expression plasmid. Cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in immunoprecipitation (IP) were examined by immunoblotting using anti-NS2 or anti-SPCS1 antibody. (D) Expression of SPCS1-myc and its deletion mutants. 293T cells were transfected with indicated plasmids. The cell lysates were examined by immunoblotting using anti-myc or anti-actin antibody. (E) Cells were co-transfected with indicated plasmids, and then lysates of transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-FLAG- or anti-myc antibody. (F) Lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates (right panel) and whole cell lysates used in IP (left panel) were examined by immunoblotting using anti-FLAG or anti-myc antibody. (G) 293T cells were transfected with indicated plasmids. 2 days posttransfection, cells were fixed and permeabilized with Triton X-100, then subjected to in situ PLA (Upper) or immunofluorescence staining (Lower) using anti-FLAG and anti-V5 antibodies. (H) Detection of the SPCS1-NS2 interaction in transfected cells using the mKG system. 293T cells were transfected by indicated pair of mKG fusion constructs. Twenty-four hours after transfection, cell were fixed and stained with DAPI, and observed under a confocal microscope.</p

Differences in induction of anti-apoptotic genes by thapsigargin in HepG2-NS5A cells compared to HepG2 control cells.

<p>We compared the induction of anti-apoptotic genes by thapsigargin in HepG2-NS5A with that in HepG2 control. Three sets of real-time PCR arrays were performed. *, HepG2-NS5A vs. HepG2 control.</p><p>Differences in induction of anti-apoptotic genes by thapsigargin in HepG2-NS5A cells compared to HepG2 control cells.</p

Mutations in the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) interferon-sensitivity determining region (ISDR) do not have impact on thapsigargin-induced apoptosis in hepatocytes.

<p>(A) Amino acid sequences of the HCV NS5A ISDRs of pCXN2-NS5A in the present study. W, wild type; I, intermediate type; M, mutant type. The sequence of HCV-J was reported by Kato et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113499#pone.0113499-Kato1" target="_blank">[42]</a>. (B), (C) No effect of HCV NS5A ISDR sequences on apoptosis was observed by thapsigargin. HepG2 cells were transfected with 0.3 µg of each vector as indicated. 24 h post-transfection cells were treated with thapsigargin at the indicated concentrations. Apoptosis was evaluated at 48 h post-transfection by APOPercentage Apoptosis Assay. Purple-red stained cells were identified as apoptotic cells by light microscopy. The number of purple-red cells/300 cells was counted. Data are expressed as mean ± standard deviation. *<i>P<0.05.</i></p

Hepatitis C virus (HCV) infection induces ER stress in hepatocytes.

<p>HCV JFH1 infection of Huh7 cells up-regulates mRNA expression of GRP78, XBP1, GADD34 and CHOP. Total cellular RNA was isolated from cells 72 h after infection with HCV. Intracellular gene expression levels of GRP78, XBP1, GADD34, CHOP and GAPDH were measured by real-time RT-PCR. The ratios of GRP78/GAPDH, XBP1/GAPDH, GADD34/GAPDH and CHOP/GAPDH are presented as induction (n-fold) relative to the levels observed in mock-infected control. Data are expressed as mean ± standard deviation. *<i>P<0.05.</i></p

Effect of SPCS1 knockdown on the production of HCV.

<p>(A) Huh7.5.1 cells were transfected with four different siRNAs targeted for SPCS1 or control siRNA at a final concentration of 15 nM, and infected with HCVcc at a multiplicity of infection (MOI) of 0.05 at 24 h post-transfection. Expression levels of endogenous SPCS1 and actin in the cells were examined by immunoblotting using anti-SPCS1 and anti-actin antibodies at 3 days post-infection. (B) Infectious titers of HCVcc in the supernatant of cells infected as above were determined at 3 days postinfection. (C) Huh-7 cells were transfected with pSilencer-SPCS1, and hygromycin B-resistant cells were selected. The SPCS1-knockdown cell line established (KD#31) and parental Huh-7 cells were subjected to immunoblotting to confirm SPCS1 knockdown. (D) KD#31 cells or parental Huh-7 cells were transfected with RNA pol I-driven full-length HCV plasmid in the presence or absence of shRNA-resistant SPCS1 expression plasmid. Expression levels of SPCS1 and actin in the cells at 5 days post-transfection were examined by immunoblotting using anti-SPCS1 and anti-actin antibodies. (E) Infectious titers of HCVcc in the supernatants of transfected SPCS1-knockdown cells or parental Huh-7 cells at 5 days post-transfection were determined.</p

Brox is required for HCV life cycle.

<p>(A) Subcellular localization of Myc-tagged TSG101 in HCV-JFH1-infected RSc cells. RSc cells transfected with 100 ng of pBj-Myc-TSG101 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014517#pone.0014517-Katoh2" target="_blank">[40]</a> were infected with HCV-JFH1. Cells were fixed 60 hrs post-infection and were then examined by confocal laser scanning microscopy as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014517#pone-0014517-g003" target="_blank">Fig. 3</a>. High magnification image of area 1 is shown. Subcellular localization of endogenous Alix in HCV-JFH1-infected RSc cells 60 hrs post-infection. Cells were stained with anti-Alix and anti-HCV Core antibodies and were examined by confocal laser scanning microscopy. High magnification images of area 1 and area 2 are shown. (B) HCV Core partially colocalizes with Brox. 293FT cells cotransfected with 100 ng of pcDNA3/core (JFH1) and 100 ng of pmGFP-Brox^WT<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014517#pone.0014517-Ichioka1" target="_blank">[27]</a> were examined by confocal laser scanning microscopy. (C) The HCV Core partially colocalizes with Brox in HCV-JFH1-infected RSc cells. RSc cells transfected with 100 ng of pmGFP-Brox^WT were infected with HCV-JFH1. Cells were fixed 60 hrs post-infection and were then examined by confocal laser scanning microscopy. (D) Inhibition of Brox mRNA expression by the shRNA-producing lentiviral vector. Real-time LightCycler RT-PCR for Brox was performed as well as for β-actin mRNA in triplicate. Each mRNA level was calculated relative to the level in RSc cells transduced with a control lentiviral vector (Con) which was assigned as 100%. (E) The levels of HCV Core in the culture supernatants from the Brox knockdown RSc cells (Broxi) 72 hrs after inoculation of HCV-JFH1 were determined by ELISA. (F) The infectivity of HCV in the culture supernatants was determined by a focus-forming assay at 48 hrs post-infection. Experiments were done in triplicate and each virus titer was calculated relative to the level in RSc cells transduced with a control lentiviral vector (Con) which was assigned as 100%. Asterisks indicate significant differences compared to the control treatment. *<i>P</i><0.05; **<i>P</i><0.01. (G) The levels of intracellular genome-length HCV-JFH1 RNA in the cells used in (E) were monitored by real-time LightCycler RT-PCR.</p