The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production

Abstract

The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Nonstructural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70. We confirmed an NS5A/HSP interaction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A. A transient transfection, luciferase-based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES-mediated translation, and small interfering RNA (siRNA)-mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion.

Conclusion: Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity.

Conflict of interest statement

Potential conflict of interest: Nothing to report.

Figures

Fig. 1

Co-immunoprecipitation of HSP40 and HSP70 with NS5A in cell-free extracts. Cell lysates from cell lines expressing either GFP or NS5A-FLAG were crosslinked with either Dithio-bismaleimidoethane or Dithiobis (succinimidylpropionate) (to improve complex stability), immunoprecipitated with anti-FLAG antibody, eluted with FLAG peptide, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and silver stained. Bands were excised and analyzed by mass spectrometry. Bands 1 and 2 were identified as HSP70 and HSP40. N, NS5AFLAG. Protein size standards are indicated on the left of the ladder in kDa. GFP control extracts were crosslinked with Dithio-bismaleimidoethane. N = 3 with reproducible results.

Fig. 2

NS5A co-immunoprecipitates with HSP40 and HSP70. Western analysis of NS5A-Flag immunoprecipitation probed for HSP40 and HSP70 demonstrates NS5A/HSP40 and NS5A/HSP70 complex formation. Input is shown at a lower exposure because of the abundance of HSP40 and HSP70 in these extracts. C, control GFP; N, NS5AFLAG. N = 2 with reproducible results.

Fig. 3

NS5A colocalizes with HSP40 and HSP70. Huh-7 cells expressing NS5AFLAG were stained with anti-Flag/anti-mouse-fluorescein isothiocyanate and either anti-HSP40 or anti-HSP70/anti-rabbit Texas red and analyzed by confocal microscopy. N = 2 with reproducible results.

Fig. 4

(A) Schematic of the IRES assay reporter construct. A 5′ cap is present upstream of Renilla Luciferase (RLuc) followed by a stop codon, the HCV IRES, and the Firefly Luciferase (FLuc). (B) IRES reporter assay demonstrates NS5A augmented HCV IRES translation that is reduced by knockdown of HSP70. Huh-7 cells were treated with the indicated siRNAs on day 1 and transfected with control or NS5A and the IRES reporter DNA plasmids on day 2. Two days after the co-transfections, Firefly and Renilla luciferase levels were measured and displayed as the ratio of Firefly/Renilla luciferase activity. Extracts were normalized to protein concentration. N = 2 with reproducible results. (C) SiRNA knockdown of HSPs. Western analysis for HSP70 and HSP40 was performed on extracts from the transfection. C, control GFP; N, NS5AFLAG. HSP40 is reduced to 52% and HSP70 to 49% by quantitative reverse transcription PCR under similar conditions (data not shown). (D) Quercetin decreases IRES activity. Experimental conditions were the same as in part B but with and without treatment with Quercetin and in the presence or absence of NS5A. N = 3 with reproducible results.

Fig. 5

(A) Quercetin treatment does not reduce viral genome replication in an HCV subgenomic replicon cell line. An Huh-7 cell line harboring a HCV subgenomic replicon was plated at low density and treated with either DMSO or Quercetin (50 µm) and RNA harvested at the hour time points indicated on the x-axis. Quantitative reverse transcription PCR was performed on DNase-treated RNA samples, and values were normalized to the 0 time point. (B) Upper panel. Quercetin lowers viral protein production in an HCV subgenomic replicon cell line. Western analysis was used to detect NS5A and actin in the same experiment performed in A. All films were exposed for the same amount of time from membranes processed at the same time. Hours are indicated at the top of the panel. D, DMSO; Q, Quercetin. N = 2 with reproducible results. Bottom panel: Ratio of NS5A signal to actin signal measured by densitometry. *P = 0.05.

Fig. 6

(A) Schematic of the intragenotype chimeric reporter HCV viral genome NRLFC used in this study. The 5′ NTR, structural regions, p7, and part of NS2 are derived from the J6CF strain, and the remaining nonstructural regions are derived from JFH-1. Renilla Luciferase (RLuc) is inserted downstream of the 5′ NTR. (B) Hepatitis C viral protein production is inhibited by Quercetin and modestly reduced by HSP70 siRNA but not HSP40 siRNA. Huh-7.5 cells were transfected with siRNA 24 hours before infection or treated with Quercetin at the time of infection. Cells were infected with NRLFC, at a multiplicity of infection of 0.1. Renilla luciferase activity was assayed on the cells at 72 hours after infection. (C) SiRNA and Quercetin are not toxic to Huh-7.5 cells. Huh-7.5 cells were treated with siRNA or Quercetin for 72 hours and MTT assays performed to determine cell viability. (D) Hepatitis C virus production is inhibited by Quercetin treatment and mildly reduced by HSP knockdown. Huh-7.5 cells were infected with NRLFC virus at a multiplicity of infection of 0.1. Cells were treated with siRNA or Quercetin. Supernatants were harvested 2 days after infection and used to infect naïve Huh-7.5 cells to determine the amount of infectious virus production. Renilla luciferase assays were performed on cells 48 hours after this second infection. N = 2 with reproducible results.