School of Molecular and Biomedical Science, University of Adelaide, Adelaide, and Centre for Cancer Biology, Adelaide, South Australia
Address reprint requests to: Michael R. Beard, Ph.D., Department of Molecular and Biomedical Science, University of Adelaide, North Terrace, Adelaide, South Australia, 5005, Australia. E-mail: email@example.com; fax: +08 8313 7532.
Potential conflict of interest: Nothing to report.
Host factors play an important role in all facets of the hepatitis C virus (HCV) life cycle and one such host factor is signal transducer and activator of transcription 3 (STAT3). The HCV core protein has been shown to directly interact with and activate STAT3, while oxidative stress generated during HCV replication in a replicon-based model also induced STAT3 activation. However, despite these findings the precise role of STAT3 in the HCV life cycle remains unknown. We have established that STAT3 is actively phosphorylated in the presence of replicating HCV. Furthermore, expression of a constitutively active form of STAT3 leads to marked increases in HCV replication, whereas, conversely, chemical inhibition and small interfering RNA (siRNA) knockdown of STAT3 leads to significant decreases in HCV RNA levels. This strongly implicates STAT3 as a proviral host factor. As STAT3 is a transcription factor, up-regulation of a distinct set of STAT3-dependent genes may create an environment that is favorable for HCV replication. However, STAT3 has recently been demonstrated to positively regulate microtubule (MT) dynamics, by way of a direct sequestration of the MT depolymerizing protein Stathmin 1 (STMN1), and we provide evidence that STAT3 may exert its effect on the HCV life cycle by way of positive regulation of MT dynamics. Conclusion: We have demonstrated that STAT3 plays a role in the life cycle of HCV and have clarified the role of STAT3 as a proviral host factor. (HEPATOLOGY 2013;58:1558–1568)
Hepatitis C virus (HCV) is a positive strand RNA virus that infects hepatocytes and can establish a chronic life-long infection resulting in progressive liver disease that can culminate in the development of hepatocellular carcinoma (HCC). Like many viruses, HCV relies on host cell factors for many facets of its life cycle. One such host factor is signal transducer and activator of transcription 3 (STAT3),[2, 3] a transcription factor that is activated by cellular stress and a wide range of cytokines. STAT3 exerts diverse cellular responses that are highly dependent on the cell type and the physiological context in which STAT3 is activated. Its importance in cell function is also highlighted by the observation that STAT3 gene knockouts are embryonically lethal in mice.
STAT3 is an 89-kDa protein that is activated by a number of growth factors and interferons (IFNs), that include: interleukin (IL)-6, cardiotrophin-1 (CT-1), leukemia inhibitory factor (LIF), epidermal growth factor (EGF), oncostatin M (OSM), and IFN-α/β. STAT3 is structurally similar to other STAT proteins and is concordantly activated by tyrosine phosphorylation (Y-705) at the carboxy terminus and serine phosphorylation (S-727) within the transactivation domain. Depending on which cytokine activates STAT3, signaling occurs through either gp130 or related receptors and tyrosine phosphorylation is most commonly mediated by way of JAK1. Activated STAT3 then follows the normal STAT paradigm, hetero/homo dimerizes, and translocates to the nucleus to activate gene transcription by way of specific DNA binding. However, while STAT3 is structurally similar to other members of the STAT family, it differs in its ability to be activated by a diverse variety of cytokines, which results in a plethora of downstream biological responses.
A role for STAT3 in the HCV life cycle has been previously suggested. It has been documented that the oxidative stress generated in HCV subgenomic replicon cell lines results in STAT3 activation. Furthermore, HCV core has been demonstrated to interact with and activate STAT3. This HCV core mediated activation of STAT3 was shown to induce expression of the STAT3-dependent genes Bcl-XL and cyclin-D1 and confirmed previous reports that constitutive STAT3 activation results in cellular transformation; an effect that may contribute to the association between chronic HCV infection and the development of HCC. Collectively, these results suggest that STAT3 is a proviral host factor; however, studies to date have been performed using Huh-7 cells constitutively harboring the HCV subgenomic replicon, with little mechanistic insight into the role of STAT3 in augmenting the HCV life cycle.
To this end, we investigated the role of activated STAT3 in the context of the full HCV life cycle, including entry, replication, and egress. We present evidence that STAT3 may enhance HCV replication by way of control of MT dynamics and we hypothesize indirectly through STAT3-dependent gene expression. These studies emphasize the need for further investigations into the role of STAT3 in the life cycle of HCV and suggest that targeting STAT3 therapeutically may limit disease progression in those with CHC. Moreover, the ability of HCV to constitutively activate STAT3 and the oncogenic nature of STAT3 suggest that HCV activation of STAT3 could be responsible in part for the increased incidence of HCC in individuals chronically infected with HCV.
Materials and Methods
Plasmids and Transfections
pRc/CMV-STAT3-C-FLAG (Jacqueline F Bromberg, Rockefeller University, NY) and pXJ40-STMN1-Myc (Dominic Chi Hiung Ng, University of Melbourne, Australia), were generous gifts. pSTAT3-Luc was purchased from Panomics (Santa Clara, CA) and transfection of all plasmids was performed using Fugene6 (Roche, Indianapolis, IN).
The human hepatoma cell lines Huh-7, Huh-7.5 (Charles Rice, Rockefeller University, NY), NNeoC-5B, and NNeo3-5B were maintained as described. Huh-7.5 cells stably expressing STAT3-C were generated using pRc/CMV-STAT3-C and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 800 μg/mL G418 (Geneticin) (Gibco, Life Technologies).
The relative luciferase activity of STAT3 promoter elements were measured using the Luciferase Assay System (Promega, Madison, WI). Cells were seeded at a density of 7 × 104 cells/well and cotransfected with pSTAT3-luc and pRL-TK the following day and 24 hours later cells were infected with HCV JFH-1 (multiplicity of infection [MOI] = 0.01). At 48 hours postinfection cell lysates were harvested as per the manufacturer's instructions and luciferase output was measured using a Glow Max Luminometer (Promega).
Infectious JFH-1[9-11] and Jc1-Myc were prepared as described. Infectivity titers were ascertained as described, with minor differences. Huh-7.5 cells were seeded into 96-well trays at 2 × 104 cells/well and cultured overnight before infection for 3 hours with viral supernatant. Cell monolayers were then washed with phosphate-buffered saline (PBS) and returned to culture for 3 days before fixation and indirect immunofluorescent labeling of HCV antigens and determination of viral titers, expressed as focus-forming units (ffu/mL).
Real-Time Polymerase Chain Reaction (PCR)
All experiments involving real-time PCR were performed using RNA extracted from cells cultured in 12-well plates. For this, Huh-7, Huh-7.5, or STAT3-C stable cells were seeded at 8 × 104/well, 24 hours prior to transfection/infection. Experiments were performed at least in triplicate. RNA extraction, first-strand complementary DNA (cDNA) synthesis, and reverse-transcription quantitative PCR (RT-qPCR) was performed as described.
Huh-7 or Huh-7.5 cells were seeded on 0.2% gelatin-coated coverslips in 24-well trays (4 × 104 cells/well) 24 hours prior to transfection/infection. Cells were fixed using methanol/acetone (1:1) for 5 minutes on ice, or with 4% paraformaldehyde for 10 minutes on ice; prior to incubation with primary antibodies for 1 hour at room temperature (RT). Cells were washed with PBS and incubated with secondary antibodies for 1 hour at RT before being mounted with Prolong Gold reagent (Invitrogen). Images were acquired with a Nikon TiE inverted fluorescence microscope (Tokyo, Japan). Mouse monoclonal anti-FLAG and rabbit polyclonal anti-FLAG were respectively obtained from Sigma (St. Louis, MO) and Rockland (Gilbertsville, PA). The rabbit monoclonal STAT3-Y705 antibody and the STAT3 rabbit polyclonal HRP were obtained from Cell Signaling (Boston, MA).
Fluorescence Resonance Energy Transfer (FRET) Analysis
FRET by acceptor photobleaching was carried out essentially as described.
Western blotting was performed as described using the following antibodies; rabbit-anti-STAT3 and phospho STAT3-Y705 (Cell Signaling), diluted 1/1,000; mouse-anti-β-actin (Sigma Aldrich, St. Louis, MO) diluted 1/10,000; mouse-anti-C-myc (clone 9E10; Roche Applied Science) diluted 1/1,000. Appropriate secondary antibodies, anti-rabbit-horseradish peroxidase (HRP) (Cell Signaling) and anti-mouse-HRP (Rockland) were diluted 1/1,000. Protein bound to antibody was then visualized by way of chemiluminescence (ECL; Amersham Bioscience, Piscataway, NJ).
Treatment of Cells With STAT3 Inhibitors
Cells were seeded in 12-well plates at a density of 7 × 104 cells/well in DMEM supplemented with 10% fetal calf serum (FCS) and returned to culture for 24 hours before fresh media was added containing STA-21 (10 μM: Biomol International, Plymouth Meeting, PA), S31-201 (20 μM: Sigma Aldrich), AG490 (10 μM: Sigma Aldrich), and a corresponding dimethyl sulfoxide (DMSO) (Sigma Aldrich) or ethanol (Sigma Aldrich) control (0.05%) for 1 hour. Cells were then infected with HCV JFH-1 (MOI = 0.01) for 3 hours, after which viral inoculums were removed, cells washed, and media replaced containing the above STAT3 inhibitors or controls. Total RNA was isolated at 24, 48, and 72 hours posttreatment for cDNA synthesis and RT-qPCR
Invitrogen Stealth STAT3 siRNA (VHS4091) and control siRNA (LoGC 12935-500) and Santa Cruz STMN1 (sc-36127) and control siRNA (sc-36869) were transfected into Huh-7.5 cells using Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions. Cells were assayed for protein knockdown at 48 hours posttransfection by way of immunoblot assay.
Densitometry analysis was performed using ImageJ as described.
Student t tests were used to analyze the distribution of two normally distributed data sets. All statistical analysis was performed using SPSS v. 10 (SPSS, Chicago, IL).
HCV Replication Increases STAT3 Activation
Previous work has shown that STAT3 is activated in Huh-7 cells that harbor HCV replication in the context of a HCV subgenomic replicon. To confirm and extend this observation we investigated STAT3 activation in the presence of HCV replication in the context of a HCV genomic replicon and the complete HCV life cycle (JFH-1). STAT3 activation requires posttranslational modifications through phosphorylation at tyrosine 705 (Y705) to be functionally active. Phosphorylation at this Y705 residue results in STAT3 dimerization and translocation to the nucleus. In the presence of both the genomic replicon (Fig. 1A) and JFH-1 infection (Fig. 1B), levels of STAT3-Y705 phosphorylation were significantly increased in the presence of HCV, in comparison to uninfected Huh-7 cells, as shown by specific detection of STAT3-Y705 by immunoblot and quantification by densitometry analysis. The presence of replicating HCV did not seem to alter total STAT3 protein within Huh-7 cells, suggesting that HCV replication does not significantly impact basal STAT3 expression (Fig. 1A,B). We next sought to determine if this HCV-dependent increase in STAT3-Y705 phosphorylation corresponded to a concomitant increase in functional STAT3 transcriptional activity. Huh-7.5 cells infected with HCV JFH-1 (48 hours) and HCV genomic replicon cells were transiently transfected with a plasmid containing a STAT3-responsive DNA element driving luciferase (pSTAT3-luc). Luciferase results were expressed as a fold change relative to the control cells (no HCV replication). HCV genomic replicon (Fig. 1C) and JFH-1 (Fig. 1D) replication significantly enhanced STAT3 luciferase output. This indicates that HCV replication induces activation of STAT3 by way of increased STAT3-Y705 phosphorylation, correlating with enhanced STAT3 transcriptional activation. Collectively, these results demonstrate that HCV replication drives activation of functional STAT3.
Activation of STAT3 Increases HCV Replication
To investigate if STAT3 activation affects the HCV life cycle, we expressed a constitutively active STAT3 molecule (PRc/CMV-STAT3-C) in Huh-7 cells harboring HCV replication. STAT3-C is a constitutively active form of STAT3 in which two cysteine residues inserted into the C-terminal loop of STAT3 allows for the formation of disulfide bonds between STAT3 monomers resulting in the formation of active STAT3 homodimers. Transient expression of STAT3-C (48 hours) resulted in a significant increase in STAT3-dependent luciferase output, indicating that this construct is functional and active in our system (Supporting Fig. 1). We then sought to express STAT3-C both transiently and stably in the context of JFH-1 infection. Transient expression of STAT3-C, followed by infection with JFH-1 (MOI = 0.01) for 24 hours resulted in a significant 2-fold increase in HCV replication (Fig. 2A). These results were then substantiated using Huh-7.5 cells stably expressing STAT3-C (Fig. 2B). JFH-1 infection of these cells for 48 hours and subsequent quantification of RNA levels by way of RT-qPCR demonstrated that stable STAT3-C expression markedly enhanced HCV replication (Fig. 2C) and supported the augmentation of HCV replication by STAT3. Furthermore, activation of STAT3 by way of exogenous cytokine treatment with LIF, a known activator of STAT3, resulted in a significant increase in STAT3 phosphorylation at Y705, as expected (Fig. 2D), while pretreatment with LIF for 24 hours prior to infection with JFH-1, resulted in a marked 2-fold increase in HCV RNA levels (Fig. 2E). These results indicate that activated STAT3 acts to either directly assist HCV replication or potentially induce the expression of specific STAT3-dependent genes that are in turn able to create an environment that is favorable for HCV replication.
Chemical Inhibition and siRNA Knockdown of STAT3 Inhibits HCV Replication
Collectively, the above experiments show that activation of STAT3 results in enhanced HCV replication. To extend these observations, the converse sets of experiments were performed using both an siRNA knockdown approach and a panel of chemical inhibitors that block STAT3 activation. To validate our knockdown approach STAT3 siRNA and a control siRNA were transfected into Huh-7 cells and total STAT3 determined by immunoblot. Despite numerous attempts, we were only able to reduce STAT3 expression by ∼50% (Fig. 3Ai). To determine the effect of STAT3 siRNA knockdown on HCV replication, Huh-7.5 cells were transfected with STAT3 siRNA or a control scrambled siRNA, and infected with HCV JFH-1. The knockdown of STAT3 with siRNA significantly decreased HCV RNA levels by ∼50% (Fig. 3Aii). These results confirm previous findings in the literature, where a genome-wide siRNA screen of Huh-7 cells infected with HCV JFH-1 revealed STAT3 as a candidate host factor involved in HCV replication.
Next we used a number of commercial STAT3 inhibitors: (1) AG490 is a JAK-2 protein tyrosine kinase (PTK) inhibitor that indirectly inhibits Y705 phosphorylation of STAT3; (2) STA-21 is a novel selective small molecule inhibitor of STAT3, which binds to the SH-2 domain of STAT3 and specifically prevents dimerization of STAT3 and DNA binding; and (3) S31-201 is a cell-permeable inhibitor of STAT3 that targets the STAT3-SH2 domain and blocks STAT3 dependent transcription. Supporting Fig. 2 outlines the STAT3 signaling cascade and demonstrates the specific points where these inhibitors exert their function.
The effects of STA-21-mediated inhibition of STAT3 on HCV replication were first investigated in an established HCV infection. HCV genomic replicon cells and JFH-1-infected Huh-7.5 cells treated with STA-21 (10 μM) for 24 hours demonstrated an approximate decrease in HCV RNA of 50% (Fig. 3B) and 70% (Fig. 3C), respectively. Given these findings, it appears that STAT3 activation, or STAT3-dependent gene expression, are involved in augmenting HCV replication at the RNA level.
We next sought to determine the effect of STAT3 inhibition with STA-21 and two other inhibitors, AG490 and S31-201, on HCV replication in the context of the complete HCV life cycle. STA-21 treatment had no significant impact on hepatocyte cell cycle progression or cell growth and was not toxic (Supporting Fig. 3); this was also observed for AG490 and S31201 (results not shown). When cells were pretreated with the STAT3 inhibitors followed by JFH-1 infection, we noted a significant decrease in HCV RNA levels by ∼70% at 24 hours (Fig. 3D). For STA-21 treatment this decrease extended to 72 hours postinfection (Supporting Fig. 4). Consistent with this decrease in HCV RNA levels, we observed a marked decrease in NS5A protein levels with STA-21 treatment (Fig. 3Eii) and demonstrated that STA-21 treatment decreases HCV RNA in a dose-response manner (Fig. 3Ei). This pretreatment scenario could indicate a block of HCV entry; however, as noted above, treatment with STA-21 of replicon cells and Huh-7.5 cells that had an established JFH-1 infection resulted in similar levels of inhibition (Fig. 3B,C), indicating that STAT3 was not acting at the level of HCV entry. Furthermore, pretreatment of Huh-7.5 cells with the STAT3 inhibitors followed by infection with JFH-1, revealed a substantial decrease in specific HCV infectivity (Fig. 4A). However, STAT3 inhibition did not affect the number or size of foci in an established infection, indicating that STAT3 does not play a role in HCV spread (Supporting Fig. 5). Concomitantly, treatment of electroporated JFH-1 Huh-7.5 cells with STA-21 and enumeration of infectious HCV in the culture supernatant revealed a significant reduction in infectious viral titers (Fig. 4B).
Taken together, these results show that inhibition of STAT3 leads to significant reduction in HCV RNA and a corresponding decrease in infectious viral titers, suggesting STAT3 plays an important role in the HCV life cycle.
STAT3 May Impact HCV Replication by Modulating Microtubule Dynamics
STAT3 could be acting to increase HCV replication either indirectly through STAT3-dependent gene expression or through STAT3 interacting with essential host cell factors. An intact MT network is essential for HCV to establish a productive infection and recently activated STAT3 has been shown to play a positive role in regulating MT dynamics, by way of sequestering the known tubulin depolymerizer protein STMN1.[21-23] We hypothesized that this ability of STAT3 to positively regulate MT activity could be a potential mechanism by which STAT3 impacts the HCV life cycle. STAT3 /STMN1 interactions have only been demonstrated in T cells and mouse embryonic fibroblasts and therefore we investigated if STAT3 and STMN1 interact in Huh-7.5 cells. Transient expression of STAT3-C and STMN1 in Huh-7.5 cells resulted in colocalization (Fig. 5A) and a physical association, as demonstrated by FRET analysis (Fig. 5B). To investigate if STA-21 disrupts the MT network we investigated the cellular distribution of α-tubulin. In STA-21-treated cells there was significant colocalization between STMN1 and α-tubulin (Fig. 5Cii), which was not observed in the control treated cells (Fig. 5Ci). These results suggest that inhibition of STAT3 enhances the ability of STMN1 to interact with α-tubulin, thereby increasing its role as a negative regulator of tubulin polymerization, which in turn disturbs MT dynamics. In support of this hypothesis, we demonstrate that STA-21 treatment induces significant disorganization of the MT network in Huh-7.5 cells (Fig. 6A). α-Tubulin displayed a dispersed punctate pattern in STA-21 treated cells, which was not observed in control treated cells, that displayed an organized MT network with long intact MTs radiating from the MT-organizing center (MTOC). If the STAT3/STMN1 interaction plays a role in HCV replication then the siRNA mediated knockdown of STMN1 should restore HCV replication in the presence of STA-21. To establish if this observation was dependent on STMN1, investigation of α-tubulin cellular distribution was performed in the presence of an siRNA knockdown of STMN1 (Fig. 6C) and STA-21 treatment (Fig. 6B). As predicted, siRNA knockdown of STMN1 rescued the effect of MT disorganization induced by STA-21. We therefore monitored JFH-1 RNA in the presence of STMN1 knockdown and STA-21 treatment and showed that under these conditions a significant but partial rebound in HCV RNA levels occurred (Fig. 6D).
This partial rescue was most likely attributed to some residual STMN1 expression and the likelihood that STAT3 impacts HCV replication through multiple mechanisms. These results indicate that STAT3 may play an important role in mediating MT dynamics to create a cellular environment favorable for HCV replication.
The number of host factors that impact the HCV life cycle continues to grow. These factors have been shown to play roles in multiple facets of the HCV life cycle, including entry, RNA replication, and egress. Early work using the HCV replicon model and more recently using a genome-wide siRNA screen have implicated STAT3 as a candidate host factor playing a role in HCV replication.[1, 2] However, to date the role of STAT3 in the HCV life cycle has been observational and this raises the question of how STAT3 exerts its effect on HCV replication, whether it is in an indirect or a direct manner.
The highly pleiotropic nature of STAT3 due in part to its ability to be activated by such a large variety of growth factors and cytokines suggests that STAT3's impact on HCV replication will be multifactorial. It is likely that in the liver, during an active HCV infection, STAT3 activation may occur by way of multiple pathways including virally induced oxidative stress, IL-6, LIF, and EGF. To this end we have shown in vitro that LIF treatment of Huh-7.5 cells markedly increases HCV RNA replication. Oxidative stress is a known activator of STAT3 and as such it is not surprising that HCV replication is capable of activating STAT3. Our study extends the work of Waris et al., and shows that replicating HCV in the form of the genomic replicon and infectious HCVcc leads to STAT3 being actively phosphorylated at the critical Y705 residue, which translates to downstream activation of the STAT3 promoter.
These results raise the question of what mechanism(s) STAT3 uses to exert its proviral effect. It is plausible that specific sets of STAT3-dependent target genes may be up-regulated in hepatocytes during HCV infection; this in turn would allow for the expression of host proteins that may assist in creating a cellular environment favorable for HCV replication. Alternatively, STAT3 dependent host cell factors, or STAT3 itself, may interact directly with viral proteins to enhance HCV replication. One such example is that STAT3 is a known transcriptional activator of VEGF, a protein capable of promoting HCV entry into hepatocytes in vivo. Thus, it is conceivable that in the HCV-infected liver STAT3 activation may facilitate HCV entry into hepatocytes. Alternatively, STAT3 may impact the host antiviral response, as STAT3 has been recently described to act as a negative regulator of the type I interferon response, by way of direct suppression of the interferon-stimulated genes OAS, PKR, and IRF7. We explored this possibility; however, we were unable to show in Huh-7 cells or primary human hepatocytes that inhibition of STAT3 activation in the presence of IFN-α resulted in increased expression of known anti-HCV ISGs (data not shown).
We have demonstrated in our study that STAT3 plays a role in HCV replication, as inhibition of STAT3 with the specific inhibitor STA-21 or siRNA-mediated knockdown of STAT3 markedly reduced HCV replication in the genomic replicon system (50%) and in the infectious JFH-1 system (70%). Moreover, we have shown that the converse set of experiments in which a constitutively active form of STAT3 is expressed both transiently and stably leads to increased HCV replication. Collectively, these results indicate that STAT3 is playing an important role in HCV RNA replication. However, as the effect of inhibition with STA-21 is greater in the context of the full life cycle of HCV, it is possible that STAT3 may also be acting at another stage of the HCV life cycle. We have shown that cell-to-cell spread of the virus is not affected by STA-21 inhibition of STAT3 and that STA-21 is still effective at inhibiting replication in an established infection. These findings suggest that STAT3 does not play a role in mediating HCV entry or spread in Huh-7.5 cells. However, in accordance with previous findings in the literature we have shown that STAT3 is likely to be involved in HCV RNA replication.
It is now becoming clear that STAT3 plays a direct and integral role in controlling the dynamics of the MT network. Activated STAT3 has been demonstrated to directly bind to, and attenuate the action of, STMN1, a known tubulin deploymerizer.[22, 23] As such, STAT3 positively regulates MT activity. The MT network and the process of MT polymerization is necessary for many viruses, including HCV, to establish a productive infection. Both HCV core and NS5A have been demonstrated to traffic along MTs and it has been suggested that HCV core integrates into the MT lattice by way of direct binding to tubulin.[28-30] Furthermore, it has been hypothesized that this association between HCV and MTs may allow the virus to exploit assembly dynamics and/or treadmilling mechanisms of MTs, which would allow for effective transport of the virus in infected cells. Given these recent findings, and our observations showing that activated STAT3 may enhance MT dynamics in Huh-7 cells and that siRNA-mediated reduction of STMN1 results in a partial reduction of HCV replication in the presence of STA-21, we hypothesize that HCV activates STAT3 directly by way of oxidative stress or indirectly by cytokines (EGF, LIF, IL-6) produced by bystander cells. This in turn may promote MT polymerization by way of STAT3-mediated attenuation of STMN1, leading to enhanced intracellular trafficking of the virus and increased RNA replication (Fig. 7). We postulate that this may be another mechanism through which HCV uses cellular processes for its own replicative strategy.
We have demonstrated that STAT3 is a proviral host cell factor and presented one potential molecular mechanism by which STAT3 promotes HCV replication, by way of positive regulation of MT dynamics. Further studies are required to ascertain other mechanisms, as it is likely that STAT3 will exert its effect in a multifaceted manner. Furthermore, the activation of STAT3 by HCV has significant clinical implications, in that activated STAT3 has been shown to play a role in HCC development. One of the inhibitors used in our study, S31-201, has also been used in vivo to show HCC regression in mice and RNAi knockdown of STAT3 has also been demonstrated to cause suppression of HCC growth.[1, 30] STAT3 inhibitors have not currently been trialed in HCC patients; however, the tyrosine protein kinase inhibitor sorafenib that is approved for HCC treatment, and has been subsequently demonstrated to inhibit STAT3. Given our findings, it is possible that therapeutic intervention of STAT3 activation may have a place in the treatment of CHC and HCV-related HCC in the future.
We thank Stanley Lemon for the use of HCV replicons and Takaji Wakita for HCV JFH-1. We also thank Kate Pilkington for flow cytometry analysis and Tom Majerczak for graphic work.