Visualization of sites of focal accumulation of ATP in cells expressing NS5A-ATeam or SGR-ATeam.

<p>(A) Huh-7 cells were transfected with NS5A-AT1.03 or NS5A-AT1.03^YEMK. Four days after transfection, the cells were analyzed using spectral imaging (405-nm excitation) of LSM510-META (Carl Zeiss). Images were processed to the CFP channel (F_CFP) and the Venus channel (F_Venus) using a linear unmixing algorithm using a reference for each spectrum. The upper panels demonstrate the signal intensity from a spectral channel with maximum intensity and represent the expression pattern of NS5A-ATeam. The lower panels are constructed from FRET ratio images (F_CFP/F_Venus) with pseudocolors. The pseudocolor scale is shown below. Scale bars, 20 µm. (B) Huh-7 cells were transfected with SGR-AT1.03^RK, SGR-AT1.03 or SGR-AT1.03^YEMK, and were analyzed in the same way as described in (A). SGR-AT1.03^YEMK -transfected cells were treated with 10 mM 2DG and 10 µg/ml OliA just before imaging and were used as a negative control. The upper panels demonstrate the intensity from a spectral channel with maximum intensity and represent the expression pattern of NS5A-ATeam processed from SGR-ATeam. The lower panels indicate square areas within FRET ratio panels magnified five-fold. Scale bars, 20 µm. (C) Cells were fixed after live-cell FRET imaging, and the same cell was analyzed by indirect immunofluorescence staining. Viral proteins were labeled with antibodies against NS5A (upper panels), NS3 (middle panels) and dsRNA (lower panels), which were detected with an Alexa Fluor 555-labeled anti-rabbit or anti-mouse antibody. ATeam panels (green) represent the expression of NS5A-ATeam processed from SGR-ATeam, and NS5A, NS3 or dsRNA panels (red) represent the immunostained signals. Enlarged views of the areas outlined by squares at a five-fold magnification are also shown. Scale bars, 20 µm.</p

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

Levels of adenosine nucleotides in HCV-infected and non-infected Huh-7 cells determined by CE-TOF MS.

<p>(A) ATP levels were reduced in HCV-infected cells. ATP, ADP, and AMP metabolites in Huh-7 cells with (gray bars) and without (open bars) HCV infection were measured by CE-TOFMS. (B) Ratios of ATP/ADP and ATP/AMP were calculated from the results depicted in (A). All data are presented as means and standard deviation (SD) values for three independent samples. Statistical differences between HCV-infected and non-infected cells were evaluated using Student's <i>t</i>-test.</p

SPCS1 forms a complex with NS2 and E2.

<p>(A) Lysates of cells, which were co-transfected with Core-p7, FLAG-NS2, and SPCS1-myc expression plasmids, were immunoprecipitated with anti-myc or anti-FLAG antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2, anti-FLAG, or anti-myc antibody. An empty plasmid was used as a negative control. (B) Cells were transfected with Core-p7 expression plasmid in the presence or absence of SPCS1-myc expression plasmid. The cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2 or anti-myc antibody. An empty plasmid was used as a negative control. The bands corresponding to immunoglobulin heavy chain are marked by an asterisk. (C) Cells were co-transfected with Core-p7 and SPCS1-myc expression plasmids. The cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2 or anti-myc antibody. (D) Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 20 nM. After 24 h, Huh7.5.1 cells were then co-transfected with FLAG-NS2 and Core-p7 expression plasmids. The lysates of transfected cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-E2 antibodies. Immunoblot analysis of whole cell lysates was also performed. Intensity of E2 bands was quantified, and the ratio of immunoprecipitated E2 to E2 in cell lysate was shown. Similar results were obtained in 2 independent experiments. (E) KD#31 cells and parental Huh-7 cells were co-transfected with FLAG-NS2, Core-p7, and NS3 expression plasmids. The lysates of transfected cells were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-FLAG, anti-E2, and anti-NS3 antibodies. Immunoblot analysis of whole cell lysates was also performed. The ratio of immunoprecipitated E2 or NS3 to E2 or NS3 in cell lysate, respectively, were shown.</p

Binary Nanoparticle Graphene Hybrid Structure-Based Highly Sensitive Biosensing Platform for Norovirus-Like Particle Detection

Nanoparticle (NP)-decorated carbon nanotubes or graphenes (GRPs) have attracted attention because of their synergic properties such as enhanced electrical conductivity, magneto-optical effect, and plasmon resonance energy transfer. These hybrid carbon nanomaterials are widely used in sensing platforms to monitor target biomolecules, gases, and chemicals. In this study, binary nanoparticles, specifically gold (Au)/magnetic nanoparticle (MNP)-decorated graphenes (GRPs), were applied in a virus-sensing platform. This hybrid material exhibited multiple functionalities, including magnetic, plasmonic, and enhanced electrical properties. The Au/MNP-GRPs were synthesized in two steps at room temperature under mild conditions and magnetically deposited on a Pt-interdigitated electrode as electrical-sensing channels. After deposition onto the electrode, the surface of Au/MNP-GRPs was conjugated with norovirus antibody to produce a norovirus-like particle (NoV-LP)-sensing platform. NoV-LPs were successfully detected by the hybrid nanomaterial-sensing platform, exhibiting high sensitivity and specificity in a concentration range from 0.01 pg to 1 ng. In this case, the limit of detection was calculated as 1.16 pg/mL. Thus, the binary nanoparticle-decorated graphene shows excellent potential as an electrical-sensing platform for biomolecules

Signal Peptidase Complex Subunit 1 Participates in the Assembly of Hepatitis C Virus through an Interaction with E2 and NS2

<div><p>Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a hydrophobic, transmembrane protein that is required not only for NS2-NS3 cleavage, but also for infectious virus production. To identify cellular factors that interact with NS2 and are important for HCV propagation, we screened a human liver cDNA library by split-ubiquitin membrane yeast two-hybrid assay using full-length NS2 as a bait, and identified signal peptidase complex subunit 1 (SPCS1), which is a component of the microsomal signal peptidase complex. Silencing of endogenous SPCS1 resulted in markedly reduced production of infectious HCV, whereas neither processing of structural proteins, cell entry, RNA replication, nor release of virus from the cells was impaired. Propagation of Japanese encephalitis virus was not affected by knockdown of SPCS1, suggesting that SPCS1 does not widely modulate the viral lifecycles of the <i>Flaviviridae</i> family. SPCS1 was found to interact with both NS2 and E2. A complex of NS2, E2, and SPCS1 was formed in cells as demonstrated by co-immunoprecipitation assays. Knockdown of SPCS1 impaired interaction of NS2 with E2. Our findings suggest that SPCS1 plays a key role in the formation of the membrane-associated NS2-E2 complex via its interaction with NS2 and E2, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates the early step of assembly of infectious particles.</p></div

A proposed model for a complex consisting of NS2, SPCS1 and E2 associated with ER membranes.

<p>A proposed model for a complex consisting of NS2, SPCS1 and E2 associated with ER membranes.</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

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