- Open Access
Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication
© Poat et al; licensee BioMed Central Ltd. 2010
- Received: 17 July 2010
- Accepted: 12 October 2010
- Published: 12 October 2010
Interferon alpha (IFN-α) binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9) to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD) of either a Stat1 (IRF9-S1C) or Stat2 (IRF9-S2C) protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE) luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.
- Replicon Cell
- Interferon Sensitive Response Element
- Trans Activate Domain
- IRF9 Protein
- Replicon Cell Line
Hepatitis C virus (HCV) infection is a major public health problem with 170 million infected individuals worldwide [1, 2]. HCV infection establishes a chronic inflammatory liver disease in over 70 percent of patients. In chronically infected individuals the disease slowly progresses over decades resulting in liver cirrhosis, hepatocellular carcinoma (HCC), and liver failure . The World Health Organization estimates that 3% of the world's population is infected with HCV and approximately three to four million new cases of HCV infection occur globally per year . Pegylated IFN-α plus ribavirin is the standard of care for the treatment of chronic HCV infection [5, 6]. Approximately one half of treated patients clear the virus infection with this regimen and as many as 20% of patients prematurely discontinue therapy due to side effects . The molecular details that determine response to treatment are not well understood.
IFN-α signal transduction is initiated by the binding of the IFN-α molecule to its surface receptor. This binding activates the receptor associated tyrosine kinases, Janus kinase 1 (Jak-1) and tyrosine kinase 2 (Tyk2), which then phosphorylate Stat1 and Stat2 proteins. Phosphorylated Stat1 and Stat2 then disassociate from the receptor and form the hetero-trimeric IFN-stimulated gene factor 3 (ISGF3) complex which then translocates into the nucleus and induces antiviral gene transcription. This cascade of biochemical reactions occurring in normal cells following IFN-α treatment is called the Jak-Stat pathway .
The mechanism of IFN-α resistance has been described to be related to several viral and host related factors [9–11]. For this purpose, we have developed multiple IFN-α resistant cell lines containing sub-genomic HCV RNA as a model to study IFN resistance. Characterization of these cell lines have revealed that Jak-Stat pathway defects give rise to the IFN-α resistant phenotype [12, 13]. A more recent analysis revealed that each of our nine resistant cell lines express a truncated IFN-α receptor 1 (IFNAR1), resulting in the functional inactivation of the IFN-α receptor (unpublished data). A fusion product of IRF9 to the TAD of either Stat1 or Stat2 was previously engineered . We show here that intracellular expression of an IRF9-Stat fusion protein in an IFN-resistant replicon cell line bypasses cellular defects and induces transcription of the genes under the control of the interferon sensitive response element (ISRE) promoter. Furthermore, we show here that intracellular expression of these constructs in an IFN-α resistant cell line containing sub-genomic HCV RNA inhibited viral replication and viral protein expression and induced HLA-1 surface expression without IFN-α treatment. These studies provide evidence that targeting the Stat1 or Stat2 proteins can induce an intracellular antiviral state independent of the IFN treatment thus providing an alternative strategy to overcome HCV resistance to standard IFN-α based therapy.
IFN-α resistant HCV replicon cell lines
IFN-α resistant cells containing sub-genomic HCV genotype 1b RNA were generated in our laboratory by prolonged treatment of the low inducer replicon cell line Con-15-3 with IFN-α as described previously . IFN sensitive S9-13 cells containing sub-genomic HCV genotype 1b HCV RNA were kindly provided by Ralf Bartenschlager, University of Heidelberg, Heidelberg, Germany. Stable cell lines, S3-GFP (IFN sensitive) and R4-GFP (IFN resistant) containing HCV genotype 2a subgenomic RNA with a green fluorescence protein (GFP) fusion in frame with HCV NS5A were prepared in our laboratory as previously described . All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, nonessential amino acids, 100 U/ml of penicillin, 100 μg/ml of streptomycin, 500 μg/ml of G418, and 5% fetal bovine serum.
Plasmid constructs and DNA transfection
Luciferase Reporter Assays
The R15-3 cell line was transfected using either IRF9, IRF9-S1C, or IRF9-S1C plus IRF9-S2C plasmid, ISRE firefly-luciferase (FL) plasmid, pRL-TK renilla-luciferase (RL) plasmid and treated with or without IFN-α (Schering, Kenilworth, NJ). At 24 hours post-transfection FL and RL activity was measured by integrating the total light emission over ten seconds with a luminometer (Luman LB9507, EG&G Bethold, Berlin, Germany). The results were normalized by dividing the FL value with the RL value for the same sample.
Nuclear Translocation Assay
R15-3 and 9-13 cells were were plated in a two well Lab-Tek chamber slide (Electron Microcopy Sciences, Hatfield, PA) at a density of 5 × 104 cells per ml. Twenty four hours later, cells were transfected with 1 μg of the respective Stat1-GFP, Stat2-GFP or IRF9-GFP plasmid DNA using Fugene-6 transfection reagent. At 48 hours post- transfection, To-Pro3 nuclear marker (Invitrogen, Molecular Probes, Oregon) was added to the samples at 1 μg/ml, and incubated for five minutes in PBS. IFN-α (1000 IU/ml) was then added to the appropriate groups. Confocal microscopy was performed using a Leica TCS SP2 confocal microscope equipped with three lasers (Leica Microsystems, Exton, PA). Optical slices were collected at 512 × 512 pixel resolution. NIH Image version 1.62 and Adobe Photoshop version 7.0 were used to assign correct colors of channels collected, including the Green Fluorescent Protein (green), To-Pro3 633 (far red), and the differential interference contrast image (DIC) (gray scale).
HLA-1 Expression by Flow Cytometry
R15-3 and 9-13 cells were transfected with IRF9, IRF9-S1C, IRF9-S2C, or IRF9-S1C plus IRF9-S2C plasmid. At 48 hours post-transfection the cultured cells were suspended in 100 μl of PBS and 20 μl of phycoerythrin conjugated mouse anti-human HLA-ABC antibody (Pharmingen, San Jose, CA) and incubated for 15 minutes at 4°C. The cells were then resuspended in 500 μl of PBS and analyzed by a BD LSR-II flow cytometer (Becton-Dickinson, Franklin Lakes, New Jersey) using BD FACS Diva software.
Ribonuclease Protection Assay (RPA) of the HCV Negative Sense Strand RNA
The antiviral properties of each IRF9 construct was evaluated by RPA detection of the HCV RNA negative sense strand utilizing a previously described method . R15-3 cells were transfected with IRF9, IRF9-S1C, IRF9-S2C, or IRF9-S1C plus IRF9-S2C plasmid in the presence or absence of IFN-α (1,000 IU/ml). At 72 hours total RNA was isolated, hybridized, and analyzed by RPA.
R15-3 cells were transfected with IRF9, IRF9-S1C, IRF9-S2C, or both IRF9-S1C, and IRF9-S2C plasmid. At 72 hours real time RT-PCR was used to quantify HCV RNA levels using a previously described method . S9-13 and R15-3 cells with and without IFN-α (1,000 IU/ml) treatment were used as positive and negative controls respectively. Values represent the mean log copies of HCV RNA per μg of cellular RNA from three experiments. Error bars represent standard deviation (SD). The student's t-test was used to compare R15-3 transfected cells to R15-3 cells plus IFN-α (1,000 IU/ml). Values < 0.05 were considered significant.
R15-3 cells were transfected with IRF9, IRF9-S1C, IRF9-S2C or both IRF9-S1C, and IRF9-S2C, and were treated with or without IFN-α (1,000 IU/ml). At 48 hours the cells were mounted onto a glass slide and stained for HCV NS3 utilizing a previously described method .
Analysis of Stat1 Phosphorylation by Co-immunoprecipitation
R15-3 and S9-13 cells were transfected with each of the plasmids listed in Fig. 1A. At 72 hours the cells were treated with or without IFN-α (1000 IU/ml). Forty-five minutes after IFN-α addition the cells were lysed by the addition of RIPA buffer with proteinase and phosphatase inhibitors (1 × PBS, 1% NP-40, 0.5% Deoxycholate, 0.1% SDS, 50 μg/ml PMSF, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 μg/ml pepstatin). The GFP primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the lysate and rotated at 4°C overnight. The next morning protein A/G plus-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added to each sample and rotated at 4°C for three hours. Western blotting was then performed using a Phospho-Stat1 Y701 (p-Stat1) primary antibody (Cell Signaling Technology, Danvers, MA) diluted (1:1,000) in blocking reagent, (0.1% tween 20, Tris buffered saline, and 5% NFDM) as described below.
Immunoblot Blot Analysis
Cell lysates were prepared from IRF9, IRF9-S1C, IRF9-S2C, or both IRF9-S1C, and IRF9-S2C transfected R15-3 cells at 72 hours post-transfection. Western blot analysis was then performed using HCV NS5B (Abcam, Cambridge, MA), p-eIF-2α (Cell Signalling Technologies, Danvers, MA) and β-actin (Cell Signalling Technologies, Danvers, MA) primary antibodies.
The viability of IRF9 fusion construct transfected R15-3 cells was evaluated by the MTT assay. At 72 hours post-transfection 100 μl of the MTT solution (Sigma, St. Louis, MO) was added to the media in each well. After three hours of incubation at 37°C the media was aspirated and 1 ml of solubilization buffer (0.1 N HCL in absolute isopropanol) was added to each well. The absorbance of each sample was then measured at 570 nm utilizing a Beckman DU Spectrophotometer after blanking. The viability was then calculated by the formula dividing the OD570 of each sample by the mean OD570 of the controls.
IFN-α independent nuclear localization of IRF9 in the R15-3 and S9-13 cells
Intracellular expression of IRF9-Stat fusion protein activates ISRE transcription
Efficient clearance of HCV replication from R15-3 cell line expressing IRF9-Stat fusion proteins
The molecular mechanisms of IFN-α resistance are unclear, however a number of studies have provided evidence that both viral and cellular factors are involved [18–23]. We provided evidence that replicon cells develop IFN-α resistance due to defective Jak-Stat signaling. Using long-term IFN-α treatment of replicon cells we have established IFN-α resistant sub-genomic HCV based stable replicon cell lines for HCV 1b and HCV 2a genotype viruses. Replication of HCV RNA in these stable cell lines remained resistant to IFN-α due to defects in Stat1 and Stat2 phosphorylation and the subsequent impairment of nuclear translocation of ISGF3. In this study, a potential therapeutic strategy against chronic HCV infection using a chimeric protein of IRF9 fused to the TAD of either the Stat1 or Stat2 molecule of the Jak-Stat signaling pathway was examined.
We showed that the IRF9 protein was expressed at intermediate levels in the R15-3 cells after transfection and efficiently localized to the nucleus, suggesting that the nuclear translocation of IRF9 protein in the R15-3 cells is not dependent upon IFN-α induced Jak-Stat signaling. Nuclear translocation of the Stat1-GFP and Stat2-GFP chimera proteins was efficient in the S9-13 cell line; however the nuclear localization of Stat1-GFP and Stat2-GFP did not occur in the R15-3 cells that possess defective Jak-Stat signaling. These results led us to examine whether the development of a chimera protein between the IRF9 and the TAD of either Stat1 or Stat2 protein could facilitate nuclear translocation and induce IFN antiviral gene expression. We showed here that intracellular expression of the IRF9-Stat1 and Stat2 fusion proteins in the R15-3 cell line activated the ISRE-luciferase promoter in a concentration dependent manner. Expression of the IRF9 protein alone did not activate the ISRE-luciferase promoter activity suggesting that IRF9 alone does not control the antiviral gene transcription induced by IFN-α. The results of this study also validate the previous finding that the fusion of the TAD of either Stat1 or Stat2 to IRF9 protein induced the ISRE promoter . The activity of IRF9-S2C was stronger than IRF9-S1C. The recombinant IRF9-Stat fusion constructs also induced HLA-1 surface expression in R15-3 cells in an IFN-α independent manner. Based on these results we speculate that intracellular expression of IRF9-S2C or IRF9-S1C in HCV infected hepatocytes may induce HLA-1 surface expression and may thus increase cytotoxic T lymphocyte (CTL) mediated viral clearance. Interestingly we observed that the intracellular expression of IRF9-S2C alone or the combination with IRF9-S1C showed stronger activation of ISRE-luciferase promoter activation than IRF9-S1C alone, indicating that Stat2 expression contributes more towards the potent antiviral activity against HCV infected hepatocytes with defective Jak-Stat signaling. On the other hand, intracellular expression of either IRF9-S1C, IRF9-S2C or the combination of both show similar results in the mobilization of HLA-1 surface expression in R15-3 cells. These results suggest that intracellular expression Stat1 fusion could contribute more towards the immunomodulatory activity in HCV infected hepatocytes with defective Jak-Stat signaling.
The antiviral properties of the IRF9-Stat fusion proteins were also examined using stable IFN-resistant cell lines of HCV 1b and 2a genotype viruses. Using the R15-3 cell line containing sub-genomic HCV genotype 1b RNA, we showed that intracellular expression of IRF9-S1C and IRF9-S2C efficiently inhibited negative strand HCV RNA levels and viral NS3 protein expression in a replicon cell line containing defective Jak-Stat signaling. This approach was also tested using IFN-α resistant HCV 2a sub-genomic GFP replicon. We showed that transient transfection of IRF9-S1C and IRF9-S2C or both inhibited GFP expression in an IFN-α independent manner. These results suggest that this antiviral strategy may be effective against different HCV genotypes. To understand the mechanism of inhibition of HCV replication in the R15-3 cell line, the protein kinase R (PKR) mediated eIF-2α phosphorylation pathway was examined by western blot analysis. Our results suggest that the mechanism of inhibition of viral replication by intracellular IRF9-Stat fusion expression is similar to that described for IFN-α and involves PKR induced eIF-2α phosphorylation .
A critical determinant in the outcome of HCV infection is the host's ability to mount an effective HCV specific CD8+ T cell response. It was also previously demonstrated that a defective Jak-Stat system contributed to impaired HLA-1 surface expression . It is therefore conceivable that the ineffective CD8+ responses of chronically infected HCV patients and IFN non-responders may in part be linked to the inability of infected hepatocytes to surface display the HLA-1: antigen complex. This impaired surface presentation may also contribute to the immune evasion observed in virally infected hepatocellular carcinoma cells. A substantial body of clinical evidence exists to support the role of dysregulated Jak-Stat signaling in IFN non-response in chronic HCV infection [26–29]. These clinical studies as well as our cell culture studies support the importance of Jak-Stat signaling in infected hepatocytes in viral persistence and response to IFN-α treatment. Using HCV cell culture models we now provide evidence that components of the Jak-Stat pathway can be engineered to circumvent defective signaling thereby inducing an antiviral state resulting in HCV eradication without IFN-α treatment. Based on these in vitro studies we propose that combination treatment using IRF9-S1C and IRF9-S2C recombinant proteins may provide a rationale for future development of an antiviral strategy to overcome IFN-α resistance by circumventing Jak-Stat cellular defects in chronically infected liver cells. We propose that liver targeted delivery of IRF9-Stat fusion protein can be used as a second line treatment in chronically infected hepatitis C patients with defective Jak-STAT signaling in an attempt to stimulate an antiviral response as well as increase HLA-1 expression in hepatocytes in an IFN-α independent manner.
This work was supported by funds received from the National Cancer Institute (CA127481, CA129776), Louisiana Board of Regents, Louisiana Cancer Research Consortium and Tulane Cancer Center. The authors acknowledge Dr. Curt Horvath, Feinberg School of Medicine, Northwestern University, Illinois, USA for the generous gift of the IRF9-STAT recombinant plasmids. The authors also acknowledge Dr. Mario Koster, Department of Gene Regulation and Differentiation, GBF-National Research Institute for Biotechnology, Braunschweig, Germany for kindly providing the STAT2-GFP construct. Ralf Bartenschlager, University of Heidelberg, Heidelberg, Germany for providing 9-13 cell line. The authors thank Dr. Astrid Engel, Dr. Sruti Chandra, and Mallory Heath for critically evaluating this manuscript.
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