Hepatitis C Virus Translation Preferentially Depends on Active RNA Replication

<div><p>Hepatitis C virus (HCV) RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome.</p> </div

Double-labeling of newly-synthesized HCV RNA and newly synthesized viral peptides in JFH1-infected Huh-7.5 cells.

<p>Huh7.5 cells were infected with HCV JFH-1 strain for 2 days, and then were labeled with Cy5-UTP and BODIPY-FL-Lys-tRNA in the presence of actinomycin D and hippuristanol, which inhibit host RNA and protein synthesis, respectively. The cells were kept in 37°C chamber supplied with CO₂ for live cell imaging on a Zeiss LSM 510 laser scanning confocal microscope. Images were taken after 10–40 minutes of chase. Newly-synthesized HCV RNA was the first to be detected (as shown in red) and was in a perinuclear pattern. Newly-translated HCV viral peptides (as shown in green) were detected at later time points, completely co-localized with the sites of RNA synthesis. No significant amount of Cy5-UTP and BODIPY-FL-Lys-tRNA labeling could be detected in naïve Huh7.5 cells (as a negative control) in the presence of actinomycin D and hippuristanol.</p

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

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 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

The proposed model of coupled replication/translation of HCV RNA.

<p>A proposed model of HCV replication-translation complex “replicasome”. As reported, HCV replication complexes are assembled at ER, and then bud into the ER lumen. Consequently, (A) HCV RNA replication is first initiated in the multi-layered vesicle structure derived from the ER membrane. (B) The newly synthesized HCV RNA is translated around the ER-derived vesicle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043600#pone.0043600-Sir1" target="_blank">[39]</a>, where there are membrane-associated ribosomes. Benzothiadiazine blocks HCV RNA transcription and therefore decreases translation. (C) The newly-synthesized HCV RNA is later transported away from ER; nocodazole inhibits this transportation. HCV RNA is then transported to Golgi-derived membrane and/or then the lipid droplet (LD) for packaging and assembly of virus particles.</p

Effect of SPCS1 knockdown on the propagation of JEV.Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 10 nM, and infected with JEV or HCVcc at an MOI of 0.05 at 24 h post-transfection.

<p>(A) Infectious titers of HCVcc in the supernatant at 3 days post-infection were determined. (B) Infectious titers of JEV in the supernatant at indicated time points were determined. (C) Expression levels of endogenous SPCS1 and actin as well as viral proteins in the cells were determined by immunoblotting using anti-SPCS1, anti-actin, anti-HCV core, and anti-JEV antibodies 3 days post-infection.</p

HCV RNA translation is dependent on the RNA transcription.

<p>(A), Mock- or Benzothiadiazine-treated Huh-N1b and Huh-Neo cells were labeled by ³⁵S-Methionine for 4 hours, and followed by immunoprecipitation with anti-NPT or anti-NS3 antibodies or sera from hepatitis C patients. The immunoprecipitates were separated by SDS-PAGE and detected by autoradiography. (B) The intracellular replicon RNA was detected by Northern blotting. (C) Huh7 cells transfected with <i>in vitro</i> transcribed Rep or the replication-defective Rep*GND RNA were metabolically labeled with ³⁵S-Methionine for 14 hours and followed by immunoprecipitation with anti-NS3 antibody. (D) The amounts of the intracellular HCV RNA in panel (C) were determined by realtime RT-PCR and Nothern blotting. The relative ratios of the HCV RNA/GAPDH mRNA in the different cells are presented. E) Structures of the bi-cistronic replicon reporter constructs used. Time course studies of the luciferase activity in cells transfected with constructs 1 and 2 (panel (F)) and constructs 3 and 4 (panel (G)) were measured by dual luciferase assay at various time points after transfection. The ratios of the FFluc/Rluc (F) or Rluc/FFluc (G) are presented. Error bars represent +/− standard deviation.</p

The translocation of newly-synthesized HCV RNA.

<p>HCV replicon cells were labeled with BrUTP (A) or ³H-Uridine (B) in the presence of actinomycin D and chased for up to 180 minutes. (A) Immunofluorescence staining with anti-BrdU and other organelle antibodies shows the colocalization of BrU-labeled HCV RNA with ER initially (30 min) and then with Golgi (180 min). (B) Fractionation of ER and Golgi by sucrose gradient. Fraction numbers and their gradient positions are noted at the bottom. ³H-Uridine-labeled RNA in the ER (fraction 4) and the Golgi (fraction 6–8) fractions were collected, and the radioactivity of ³H-Uridine-labeled RNA was counted. Immunoblotting of ER and Golgi makers demonstrates the separation of ER and Golgi by sucrose gradient fractionation.</p