Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production

Abstract

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.

Fig. 1.

Expression of TAP-NS3/4A and identification of associated host factors. (A) Schematic representation of TAP-NS3/4A. 293EcR cells were transfected with NS3/4A-, TAP-, or TAP-NS3/4A-expressing plasmids. Twenty-four hours posttransfection, cell extracts were prepared and subjected to Western analysis using polyclonal rabbit antibodies that nonspecifically recognize the protein A moiety of the TAP tag and allow the visualization of TAP and TAP-NS3 proteins (B), as well as anti-NS3, anti-MAVS, and anti-GAPDH antibodies (C). The asterisks indicate the NS3 degradation products detected with the anti-NS3 antibodies with different exposures (expo.). (D) Cells were treated as described for panel B in the presence of DMSO or 2 μM the NS3 protease inhibitor BILN2061. Cell extracts were analyzed by Western blotting (WB) using anti-NS3 antibodies and subjected to TAP. Resulting eluates were analyzed by Western blotting using anti-NS3 antibodies and silver staining. (E) The host factors that were copurified with TAP-NS3/4A and absent from the TAP tag control eluate were identified using mass spectrometry.

Fig. 2.

YB-1 specifically interacts with HCV NS3 when expressed transiently or in the context of JFH-1 expression. (A) 293T cells were transfected with Flag-NS3/4A-expressing plasmid. Forty-eight hours posttransfection, cell extracts were prepared and subjected to immunoprecipitation (IP) using an anti-Flag-coupled resin. Resulting eluates as well as cell extracts were analyzed by Western blotting using anti-NS3, anti-YB-1, anti-hnRNP A1, anti-hnRNP U, anti-hnRNP A1, and anti-RHA antibodies. Anti-S6 was used as a loading control for cell extracts. (B) 293T cells were cotransfected with plasmids encoding Flag-YB-1 and several YFP-tagged HCV nonstructural proteins. Forty-eight hours posttransfection, cell extracts were prepared and subjected to immunoprecipitation using an anti-Flag-coupled resin. Resulting eluates as well as cell extracts were analyzed by Western blotting using anti-GFP antibodies. (C) Huh7.5 cells were transfected with control DNA (Mock) or JFH-1-expressing plasmid. Three days posttransfection, cell extracts were prepared and subjected to immunoprecipitation directed against the HA epitope and endogenous proteins YB-1 or RHA. Immune complexes were analyzed by Western blotting using anti-RHA, anti-YB-1, and anti-NS3 antibodies. IgG indicates nonspecific labeling due to the presence of antibodies in the loaded samples.

Fig. 3.

YB-1 expression levels are important for HCV life cycle. (A) Huh7.5 cells were infected with lentiviruses that express shRNA silencing YB-1 (shYB-1) and RHA (shRHA). As a control, a nontarget shRNA (shNT) was used. Transduced cells were selected with puromycin, and knockdown efficiencies for YB-1 and RHA were confirmed by Western blotting. (B) shNT-, shRHA-, and shYB-1-expressing Huh7.5 cells were infected with JFH-1. Five days later, actin and JFH-1 RNAs were quantitated using qRT-PCR. HCV RNA levels were normalized with actin RNA content and arbitrarily set to 1 for the JFH-1-plus-shNT condition. Error bars represent standard deviations from biological triplicates. (C) Transduced Huh7.5 cells were transfected with JFH-1-expressing plasmids, collected each day, and analyzed for their content of actin and HCV RNA as described for panel B. The normalized levels of HCV RNA 1 day posttransfection were arbitrarily set to 1 for each condition to monitor viral RNA amplification. Error bars represent standard deviations from biological duplicates. (D) Huh7.5 cells were treated as described for panel C and analyzed 4 days posttransfection for their content in core protein, YB-1, and actin by Western blotting. (E) Genotype 1b subgenomic replicon-containing 9-13 cells were transduced with shRNA-expressing lentiviruses and were analyzed 5 days later for their content of HCV RNA using qRT-PCR. Error bars represent standard deviations from biological triplicates. (F and G) Huh7 cells that stably express a luciferase-producing Con1 subgenomic replicon were transduced with shRNA-expressing lentiviruses. Luciferase and MTT assays were performed each day following transduction as readouts of HCV replication (F) and cell viability (G), respectively. As a control, cells were transduced with shNT lentiviruses in the presence of BILN2061. Error bars represent standard deviations from four biological replicates.

Fig. 4.

Characterization of the ribozyme-based JFH-1 infectious system. (A) Schematic representation of the wild-type construct as well as assembly (del153-167)- and replication (GND)-deficient JFH-1 DNA constructs. (B) Huh7.5 cells were transfected with the different JFH-1 constructs represented. Seventy-two hours posttransfection, cell extracts were prepared and analyzed by Western blotting using anti-NS3, anti-NS5A, anti-core protein, and anti-actin antibodies. (C) Culture media from transfected cells described for panel B were used to infect naive Huh7.5 cells. After 4 days, cellular RNA was purified and actin and JFH-1 RNAs were determined using qRT-PCR. HCV RNA levels were normalized with actin RNA content and arbitrarily set to 1 for wild-type JFH-1. As a control for the complete inhibition of HCV replication, cells were cultured with 2 μM BILN2061. Error bars represent standard deviations from biological triplicates. (D) Huh7.5 cells were transfected as described for panel B. Transfected cells then were collected each day and analyzed for HCV RNA levels as described for panel C in a 6-day kinetic study. The normalized HCV RNA levels at day 1 posttransfection were arbitrarily set to 1 for each construct to monitor viral RNA amplification. Error bars represent standard deviations from biological duplicates.

Fig. 5.

YB-1 associates with HCV RNA. (A) Huh7.5 cells were transfected with control and JFH-1-expressing plasmid. Seventy-two hours posttransfection, cell extracts were prepared and subjected to immunoprecipitation directed against YB-1, RHA, hnRNP U, and RNA polymerase II (Pol II) endogenous proteins. Antibodies against the HA epitope were used for negative-control immunoprecipitations. Immune complexes were analyzed by Western blotting using anti-NS3, anti-YB-1, anti-RHA, anti-hnRNP U, and anti-Pol II antibodies. IgG indicates nonspecific labeling due to the antibodies loaded on the gel. (B) Immune complexes from panel A were subjected to RNA extraction, and HCV RNA from each immunoprecipitate was quantitated using qRT-PCR. Controls without reverse transcriptase (RT) were included to monitor for plasmid DNA contamination. (C) Huh7.5 cells were cotransfected with plasmids encoding JFH-1 and Flag-tagged YB-1. Seventy-two hours posttransfection, cell extracts were prepared and subjected to immunoprecipitation using an anti-Flag-coupled resin. Resulting eluates as well as cell extracts were analyzed by Western blotting using anti-Flag and anti-actin antibodies. RNA was purified from cell extracts (D) and immunoprecipitates (E), and HCV RNA was quantitated using qRT-PCR. All error bars represent standard deviations from three independent experiments.

Fig. 6.

HCV induces YB-1 relocalization to core-containing lipid droplets. Control and JFH-1-expressing Huh7.5 cells were fixed and probed with rabbit anti-YB-1 (A), anti-RHA (B) (green), and mouse anti-core (red) antibodies. Nuclei and lipid droplets were stained with Hoechst dye (white) and LipidTOX (blue), respectively. Yellow arrows, core-containing ring-like structures; white arrows, core-free large structures. Unmerged and merged higher-magnification images of the red selected area are shown. The white asterisks indicate untransfected cells.

Fig. 7.

Core protein is necessary for HCV-induced YB-1 relocalization to lipid droplets. (A) Huh7.5 and subgenomic replicon-containing 9-13 cells were cultured in the presence of DMSO or 2 μM BILN2061 for 5 days. Cell extracts were prepared and analyzed by Western blotting using anti-NS3, anti-YB-1, anti-MAVS, and anti-GAPDH antibodies. GAPDH was used as a loading control. (B) 9-13 cells were grown on coverslips as described for panel A. After 5 days of culture, cells were fixed and probed with rabbit anti-YB-1 (green) and mouse anti-NS3 (red) antibodies. (C) Huh7.5 cells were transfected with plasmids encoding either wild-type JFH-1 or the del153-167 JFH-1 mutant. Following 4 days of culture, cells were fixed and probed with rabbit anti-YB-1 (green) and mouse anti-NS3 (red) antibodies. Nuclei and lipid droplets were stained with Hoechst dye (white) and LipidTOX (blue), respectively. Unmerged and merged higher-magnification images of the red selected area are shown. Yellow arrows indicate YB-1/NS3 colocalization foci. (D) Huh7.5 cells were treated as described for panel C in the presence of 1 μM BILN2061 in the culture media.

Fig. 8.

YB-1 localization phenotype of various replication- and assembly-defective JFH-1 mutants. A schematic representation of each JFH-1 mutant is shown above the corresponding panel. JFH-1 mutant-expressing Huh7.5 cells were fixed and probed with rabbit anti-YB-1 (green) and mouse anti-core (red) antibodies. Nuclei and lipid droplets were stained with Hoechst (white) and LipidTox (blue) dyes, respectively. Merged images are shown.

Fig. 9.

YB-1 expression knockdown significantly increases the production of infectious viral particles. shNT- and shYB-1-expressing Huh7.5 cells were transfected with wild-type and mutant JFH-1-expressing plasmids. (A) Two days posttransfection, cells were collected and analyzed for their content in YB-1 and HCV RNA using Western blotting and qRT-PCR, respectively. The corresponding cell supernatants were collected, analyzed for their content in HCV RNA (B), and used to infect naive Huh7.5 cells (C). Three days postinfection, naive cells were collected and analyzed for their HCV NS3 and RNA content using Western blotting and qRT-PCR, respectively. HCV RNA levels were normalized with actin RNA content and arbitrarily set to 1 for the JFH-1-plus-shNT condition. All error bars represent standard deviations from biological triplicates.