A host YB-1 ribonucleoprotein complex is hijacked by hepatitis C virus for the control of NS3-dependent particle production

Abstract

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.

Fig 1

A combined MS-RNAi screening approach identifies new HCV replication regulators. (A) Decision tree for the identification of YB-1 protein partners that regulate HCV particle production. (B) Huh7 cells were transfected with Flag–YB-1-expressing or control vectors. At 48 h posttransfection, cell extracts were prepared and subjected to immunoprecipitation (IP) with anti-Flag antibody-coupled resin. Cell extracts were analyzed by Western blotting with anti-YB-1, anti-Flag, and anti-actin antibodies. The content of the immunoprecipitates was analyzed by silver staining, attesting to the specificity of the immunoprecipitation and of the copurification of YB-1 partners. (C) Schematic representation of the reporter genotype 1b HCV subgenomic replicon stably expressed by the Huh7-Con1-Fluc cell line. This replicon expresses the Fluc and G418 resistance product in a bicistronic manner under the control of the HCV internal ribosome entry site (IRES), as well as the NS3-NS5B replication unit downstream encephalomyocarditis virus (EMCV) IRES. With the absence of structural proteins, this virus does produce particles and replicates only within the cell. (D) Plate map and assay design used for RNAi screening. Huh7-Con1-Fluc cells were seeded into the wells of 96-well plates and transduced with shRNA-expressing lentiviruses. The right and left columns were used exclusively for controls. Fluc and alamarBlue assays were performed 4 days postransduction. (E) TIGR (The Institute for Genomic Research) MultiExperimentViewer (TMeV) representation of the selected shRNA hits. Each column represents the results of the RNA screening for one shRNA hit with the indicated targeted protein at the top.

Fig 2

Interaction and validation of YB-1 partner hits. (A, B) Huh7.5 cells stably expressing JFH-1 were transfected with the indicated Flag vectors. At 48 h posttransfection, cell extracts were prepared and subjected to immunoprecipitation (IP) with anti-Flag antibody-coupled resin. Resulting eluates, as well as cell extracts, were analyzed by Western blotting with anti-Flag, anti-NS3, anti-DDX3, anti-DDX6, anti-C1QBP, anti-IGF2BP2, anti-LARP1, anti-ILF2, anti-ILF3, anti-PABPN1, anti-DHX9, and anti-actin antibodies. Stau1 and IGF2BP3 MS hits were also detected in the Flag–YB-1 immunoprecipitate (data not shown). (C) Immunoprecipitation of endogenous proteins YB-1, DDX3, DDX6, IGF2BP2, LARP-1, ILF2, RHA, PABPN1, and ILF3, followed by Western blotting detection of endogenous YB-1 (Reciprocal Co-Ip) and Western blotting detection of other endogenous proteins of the YB-1 RNP complex, i.e., DDX3, DDX6, IGF2BP2, LARP-1, ILF2, RHA, PABPN1, and ILF3. Immunoprecipitation of FLAG protein and endogenous polymerase II (POLII) was used as a negative control. Ctrl −, negative control; Ab, antibody; arrow, specific protein; *, heavy chain of immunoglobulin; **, nonspecific protein.

Fig 3

Assessment of the silencing efficiency of shRNAs and functional validation of YB-1 partner hits. (A to K) Two to 5 shRNAs per hit were selected from the TRC shRNA lentiviral library (Sigma-Aldrich) of the IRIC High-Throughput Screening Core Facility. ShRNA-expressing lentiviruses were produced in 293T cells and used for the transduction of Huh7, Huh7.5, or 293T cells. The following day, puromycin was added to the cells. Seventy-two hours later, cells were collected and lysed and their contents were analyzed for the indicated host factors by Western blotting with antibodies against YBX2 (A), C1QBP (B), DDX5 (C), DDX6 (D), DDX21 (E), DDX28 (F), DHX30 (G), hnRNPG-T (H), IGF2BP2 (I), PABPN1 and LARP1 (J), and ZC3HAV1 (K) indicated in Table S1 in the supplemental material. The shRNAs in bold are those that were selected for further experiments. (L) Huh7-Con1-Fluc cells were transduced with shRNA-expressing lentiviruses at an MOI of 5. Fluc and MTT assays were performed 3 and 4 days postransduction as readouts of HCV replication and cell viability, respectively. Huh7.5 cells were transduced with shRNA-expressing lentivirus and infected the day after with J6/JFH(p7-Rluc2A) reporter HCVs. The levels of HCV infection were monitored by measuring Rluc activity at 3 days postinfection. As a control for virus replication, cells were treated with 1 μM HCV protease inhibitor BILN 2061. Nontransduced cells control for potential off-target effects of the lentiviruses on cell viability and HCV replication. Statistically significant differences are indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to the shNT-expressing cell condition).

Fig 4

HCV induces the relocalization of DDX3, DDX6, IGF2BP2, LARP1, and C1QBP to core-containing LDs. (A to G) JFH-1-expressing Huh7.5 cells were fixed and probed with antibodies that recognize the indicated host and viral proteins. Nuclei and LDs were stained with Hoechst dye and LipidTOX, respectively. Unmerged and merged higher-magnification images of the boxed areas are shown. Yellow and white arrows indicate JFH-1-expressing cells and uninfected cells, respectively. (H) Huh7.5 cells were transfected with JFH-1 and the control vector. Five days later, cells were treated with 200 μM BSA-complexed oleic acid. Sixteen hours later, cells were collected and LDs were purified with a sucrose-based flotation assay. Aliquots of lysed cells and the LD fraction were analyzed by Western blotting with anti-ADRP, anti-core, anti-NS3, anti-YB-1, anti-DDX3, anti-DDX6, anti-IGF2BP2, anti-C1QBP, anti-hnRNG-T, and anti-actin antibodies.

Fig 5

DDX3 and IGF2BP2 are corecruited with YB-1 to core-containing LDs. Huh7.5 cells, which stably express JFH-1, were transfected with eYFP-tagged YB-1, fixed 2 days later, and then probed with antibodies directed against core and DDX3 (A) or core and IGF2BP2 (B). Nuclei in panel A were stained with Hoechst. YB-1 was detected via the intrinsic fluorescence emitted by its eYFP tag.

Fig 6

Knockdown of YB-1 partners significantly stimulates the production of infectious viral particles. Huh7.5 cells transduced with shRNA-expressing lentiviruses were transfected with a JFH-1-expressing plasmid. (A) Three days posttransfection, cells were collected and lysed while released viral particles were purified from culture medium. Cell extracts and lysed viruses were analyzed for their core contents by Western blotting. (B) Virus-containing culture media from panel A were used to infect naive Huh7.5 cells. Three days postinfection, cells were collected and analyzed for their HCV RNA content by qRT-PCR. HCV RNA levels were normalized to actin RNA content and arbitrarily set to 1 for the JFH-1-plus-shNT condition. Statistically significant differences are indicated. *, P < 0.05; NS, not significant (compared to the shNT-expressing cell condition).

Fig 7

YB-1 RNP modulates the release and egress of HCV particles but not their assembly. Huh7.5 cells transduced with shRNA-expressing lentiviruses were transfected with a JFH-1-expressing plasmid. (A) Three days posttransfection, cells and culture media were collected. Intracellular viruses were prepared by freeze-thaw-based cell lysis. Virus-containing culture media (extracellular) and cell extracts (intracellular) were used to infect naive Huh7.5 cells. Three days postinfection, cells were collected and analyzed for their HCV RNA content by qRT-PCR. HCV RNA levels for intracellular and extracellular infectivities were normalized to HPRT1 RNA content and arbitrarily set to 1 for each JFH-1-plus-shNT condition. Statistically significant differences are indicated. *, P < 0.05; **, P < 0.01 (compared to the corresponding intracellular infectivity condition). (B) Huh7.5 cells were cotransfected with JFH-1- and Flag–YB-1-expressing plasmids. Three days later, an aliquot of transfected cells was analyzed for its Flag–YB-1 and HCV contents by Western blotting with anti-Flag and anti-NS3 antibodies (left side). Extracellular and intracellular viruses (prepared as in panel A) were used for infection. Infected cells were analyzed as in panel A, except that actin RNA was used for normalization (lower right side).

Fig 8

YB-1 knockdown decreases the association of NS3, DDX3, DDX6, and IGF2BP2 with LDs. Huh7.5 cells transduced with shRNA-expressing lentiviruses were transfected with a JFH-1-expressing plasmid. (A) Three days later, cells were collected and LDs were purified with a sucrose-based flotation assay. The LD fractions were analyzed by Western blotting with anti-ADRP, anti-core, anti-NS3, anti-DDX3, anti-DDX6, and anti-IGF2BP2 antibodies. (B) Cells were treated as in panel A, fixed, and probed with anti-NS3 and anti-core antibodies. Nuclei and LDs were stained with Hoechst dye and LipidTOX, respectively. Cells were analyzed by laser scanning microscopy. (C) Percentages of colocalization of NS3 or core with LDs were calculated for each condition with MetaMorph image analysis software and plotted.

Fig 9

The virus production of the JFH-1 NS3 Q221L mutant is partially unresponsive to the knockdown of YB-1 RNP factors. (A) Huh7.5 cells were transfected with constructs encoding wild-type JFH-1 and mutant JFH-1 clones. Three days posttransfection, cells and released viral particles were collected. Cells were lysed and analyzed for their NS3, core, and actin contents by Western blotting (top). Virus-containing culture media from panel A were used to infect naive Huh7.5 cells. Three days postinfection, cells were collected and analyzed for their HCV RNA content by qRT-PCR (bottom). HCV RNA levels were normalized to actin RNA content and arbitrarily set to 1 for the wild-type (wt) JFH-1 condition. Statistically significant differences are indicated. *, P < 0.01; **, P < 0.0001 (compared to wild-type JFH-1). (B) Huh7.5 cells transduced with shRNA-expressing lentiviruses were transfected with a wild-type, NS2 Q199R, or NS3 Q221L JFH-1-expressing plasmid. Three days posttransfection, cells and culture media were collected. Cells expressing wild-type JFH-1 were lysed and analyzed for knockdown efficiency and core expression by Western blotting with anti-YB-1, anti-DDX6, anti-C1QBP, anti-IGF2BP2, anti-LARP1, anti-core, and anti-actin antibodies. (C) Virus-containing culture media were used to infect naive Huh7.5 cells. Three days postinfection, cells were collected and analyzed for their HCV RNA content by qRT-PCR. HCV RNA levels were normalized to actin RNA content. In order to separately compare the effects of the shRNAs on the JFH-1 clones, the relative HCV infectivity under shNT conditions for each JFH-1 construct was used as an individual reference and arbitrarily set to 1. (D) Huh7.5 cells were transfected with wild-type and NS3 Q221L JFH-1-expressing plasmids. Three days posttransfection, cell extracts were prepared and subjected to immunoprecipitation (IP) with antibodies directed against YB-1. Anti-hemagglutinin antibody immunoprecipitation was used as a control to monitor for nonspecific binding of lysate protein to the antibodies and/or the resin. Resulting eluates (right side) and cell extracts (left side) were analyzed by Western blotting with anti-YB-1, anti-NS3, anti-IGF2BP2, and anti-actin antibodies.