Interferon Regulatory Factor 3-Independent Double-
http://www.100md.com
病菌学杂志 2005年第5期
Department of Biological Sciences, Boehringer Ingelheim Canada Ltd., Research and Development, Laval, Quebec, Canada
ABSTRACT
The treatment of human embryonic kidney 293 cells harboring a hepatitis C virus (HCV) subgenomic replicon with the double-stranded RNA (dsRNA) mimic poly(I · C) inhibits HCV RNA replication through an undefined mechanism. Interferon regulatory factor 3 (IRF 3) has been widely postulated to mediate various antiviral responses, and its role in mediating the response to dsRNA in 293 cells was examined. Treating the cells with dsRNA did not induce IRF-3 activation, as measured by nuclear localization or the induction of reporter genes. Moreover, the expression of a dominant negative form of IRF-3 did not affect either colony formation upon transfection of subgenomic replicon RNA or the inhibition of the HCV replicon by dsRNA. Our results suggest that the inhibition of HCV RNA replication by poly(I · C) in 293 cells is independent of IRF-3 activation.
TEXT
The establishment of the hepatitis C virus (HCV) subgenomic replicon system in Huh-7 cells (4, 15, 18-20) has permitted a preliminary investigation of the interactions between HCV and host cells. We have established a similar HCV subgenomic replication system in human embryonic kidney (HEK) 293 cells (2, 25). In contrast to what occurs in Huh-7 cells, HCV RNA replication can be inhibited in 293 cells upon treatment with the double-stranded RNA (dsRNA) mimic poly(I · C) (50% inhibitory concentration [IC50], 77 μg/ml].
dsRNA, a by-product of virus replication, is believed to activate the innate immune system and mediate the production of alpha/beta interferon (IFN-/?) and other cytokines (10). dsRNA and poly(I · C) are candidate ligands for Toll-like receptor 3 (TLR-3) (1, 10, 21). Engagement of poly(I · C) with the receptor is proposed to activate a signaling cascade that is MyD88 independent and involves the recruitment of Toll or interleukin 1 receptor domain-containing, adaptor-inducing IFN-? (TRIF) (22, 26, 27), which in turn is postulated to activate various signaling mediators, including IB kinase and TANK-binding kinase 1. These kinases phosphorylate and activate IFN-regulatory factor 3 (IRF-3) (8, 22, 24), which has emerged as a critical mediator of antiviral responses (23). The role of IRF-3 in the induction of antiviral responses by dsRNA remains to be fully elucidated. HEK 293 cells do not normally express TLR-3, and dsRNA can activate IRF-3 in these cells when TLR-3 is ectopically expressed (1, 14). HEK 293 cells harboring the HCV replicon that we had generated are sensitive to dsRNA (2), and they provided a unique tool to examine the role of IRF-3 in mediating the inhibition of HCV RNA replication.
dsRNA does not induce the nuclear localization of IRF-3. The nuclear localization of IRF-3 was examined with a green fluorescent protein (GFP)-IRF-3 construct that fused the GFP coding sequence to the N-terminal portion of IRF-3 in a mammalian expression vector. The GFP-IRF-3 construct was transfected into 293 and Huh-7 cells using FuGene-6 (Roche). The cells were then treated either with 20 hemagglutination units (HAU) of Sendai virus (SenV) per ml (17) or with a dose of two dsRNA mimics, poly(I · C) and poly(U · A), ranging from 50 to 500 μg per ml and then fixed, permeabilized, and stained with DAPI (4',6'-diamidino-2-phenylindole). Transfection of dsRNA into the cells was unnecessary, as adding poly(I · C) to cell culture media strongly inhibits HCV RNA replication in 293 cells (2). In untreated cells, IRF-3 localized primarily to the cytoplasm. As expected, treating S22.3 and LIP3 cells (Huh-7 and 293 cells that harbor the subgenomic replicon, respectively) with SenV and/or poly(I · C) failed to induce the nuclear localization of IRF-3 in either cell line, consistent with the known suppression of IRF-3 activation by the NS3/NS4A protease (9) (Fig. 1A). Treating na?ve 293 and Huh-7 cells with SenV induced clear nuclear localization of IRF-3, suggesting that the activation of IRF-3 is not defective in either cell type, even in Huh-7 cells that do not respond to treatment with dsRNA in inhibiting the replicon RNA (Fig. 1B and C). In contrast to the results of treatment with SenV, na?ve cells treated with 50 to 500 μg of dsRNA per ml showed no effect on IRF-3 localization; extending the treatment with 250 μg of dsRNA per ml for up to 72 h also had no effect on the nuclear localization of IRF-3 [Fig. 1B and C depict representative poly(I · C) concentrations and time points]. The activation of endogenous IRF-3 was also examined using indirect immunofluorescence staining and was found to correlate to the activation pattern of the GFP-IRF-3 reporter. Only SenV, and not poly(I · C), treatment induced the nuclear localization of endogenous IRF-3 (data not shown). Therefore, it appears that dsRNA does not induce the activation of IRF-3 in either na?ve or replicon 293 and Huh-7 cell lines.
Transactivation of the ISRE promoter by IRF-3. The induction of a luciferase reporter gene controlled by IFN-stimulated response elements (ISRE) was used as an alternative method to assess the activation of IRF-3 (3, 5). Cells were transfected with an IRF-3-regulated luciferase reporter containing five ISRE upstream of a firefly luciferase gene (pISRE) and a constitutive Renilla luciferase reporter (pRL-TK; Promega), which served as an internal control. Luciferase readings (Dual-Glo luciferase; Promega) revealed that cells treated with 5 to 50 HAU of SenV per ml induced an approximately 35-fold activation (Fig. 2A); however, no significant activation of the luciferase reporter was observed in cells treated with a dose of dsRNA ranging from 50 to 500 μg/ml. Similarly, a firefly luciferase gene reporter under the control of the IRF-3-regulated RANTES promoter (11, 16) demonstrated that treatment with dsRNA (50 to 500 μg/ml) also did not induce measurable activation relative to the activation induced by SenV treatment (Fig. 2B). Therefore, consistent with the subcellular-localization experiments, dsRNA failed to induce the transactivation of IRF-3-responsive genes, suggesting that IRF-3 is not an activated downstream target of dsRNA in these cells. Na?ve, TLR-3-negative cells were used in the above-described experiments, which may account for the difference in the levels of activation of IRF-3 in response to dsRNA treatment that was previously reported. Either TLR-3-positive cells, ectopic overexpression of TLR-3, or introduction of dsRNA into the intracellular milieu through transfection was employed (1, 6, 14).
Generation of stable cell lines expressing dominant negative IRF-3. Deleting the DNA binding domain of IRF-3 (17) generates a well-characterized dominant negative form, IRF-3 N, which we used to examine the role of IRF-3 in mediating the dsRNA-induced inhibition of HCV RNA replication. Flag-tagged IRF-3 N was constructed and transfected (Lipofectamine 2000; Invitrogen) into 293/FRT cells (Flp-In system; Invitrogen). A stable cell line, 2IN, was generated by selecting cells with 0.2 mg of hygromycin per ml. The stable expression of truncated IRF-3 was confirmed in 2IN cells by using IRF-3- and Flag-specific Western blotting (Fig. 3A). The level of expression of IRF-3 N in these cells was significantly higher than that of the endogenous protein (Fig. 3A), a prerequisite to eliciting the dominant negative effect (compare lanes 293 and 2IN-4).
The dominant negative phenotype of IRF-3 N was confirmed by using the ISRE and RANTES luciferase reporters in na?ve 293, LIP3, and in 2IN cells. Transfecting the three cell lines as described above and treating them with 5 to 50 HAU of SenV per ml revealed that, relative to the induction of luciferase activity in na?ve 293 cells, IRF-3 activation was suppressed in LIP3 replicon cells due to the presence of the NS3/NS4A protease (9) and was diminished in 2IN cells due to the expression of IRF-3 N (Fig. 3B). This result confirms that the dominant negative form interferes with the function of endogenous IRF-3, as previously reported (12, 17). Basal luciferase activities were comparable in luciferase reporter-transfected cells expressing IRF-3 N (2,776 ± 95 cps) and wild-type IRF-3 (1,895 ± 274 cps), indicating that the transfection procedure did not induce the activation of IRF-3.
Dominant negative IRF-3 does not affect HCV RNA replication. Transfection into na?ve 293 cells of total RNA obtained from 293 cells harboring the replicon generates approximately 7.5 x 103 neomycin-resistant colonies per μg of replicon RNA (2). In order to assess the effect of the stable expression of dominant negative IRF-3 on the efficiency of colony formation, 20 μg of total RNA isolated from 293Rep17 cells containing an estimated 107 replicon copies per μg was transfected as described previously (2) into 2PD cells (equivalent to na?ve 293 cells) (stable cells were generated by transfecting the empty Flp-In vector), 2IW cells (293 cells stably overexpressing wild-type IRF-3), or 2IN cells. Selecting the cells with 0.6 μg of neomycin per ml, staining the cells with crystal violet, and quantifying the resulting colonies indicated that there was no measurable effect on the number of replicon colonies generated with 2IN cells compared to the number of 293 or 2IW cells in repeated experiments (Fig. 4A). Therefore, IRF-3 does not appear to mediate an antiviral response expected to be activated by the presence of replicon RNA in the cells.
Several replicon-containing 2IN cells were expanded into cell lines, termed 2NR cells, and the maintenance of the IRF-3 N phenotype in these expanded cell lines was confirmed (data not shown).
IRF-3 does not modulate HCV RNA inhibition by dsRNA. dsRNA potently inhibits HCV RNA replication in 293 cells harboring the replicon (IC50, 77 μg/ml) (2). Treating na?ve 293, LIP3, and 2NR cells (replicon 293 cells stably expressing IRF-3 N) with a 3.9- to 1,000-μg/ml dose of poly(I · C) for 72 h revealed that the stable overexpression of dominant negative IRF-3 N did not desensitize the cells and shift the IC50 of dsRNA. The levels of inhibition of HCV RNA replication were comparable in LIP3 and 2NR cells, yielding IC50s of 63.5 ± 8.9 μg/ml for LIP3 cells and 44.7 ± 3.1 μg/ml for 2NR cells, as measured by TaqMan real-time reverse transcription-PCR (2) (Fig. 4 B). Therefore, IRF-3 does not appear to mediate an essential role in the inhibition of HCV replicons by dsRNA.
Our combined data examining IRF-3 subcellular localization, transactivation, and dominant negative suppression indicate that IRF-3 is not activated by topical dsRNA treatment and that the inhibitory response to dsRNA on HCV RNA replication observed in 293 cells is IRF-3 independent.
A recent report by Edelmann et al. (7) questioned the role of TLR-3 as the sole recognition element mediating antiviral responses to dsRNA, and a study by Hoebe et al. (13) reported TLR-3/TRIF axis-independent antiviral responses to dsRNA. Therefore, it appears that in addition to the reported TLR-3/TRIF/IRF-3 antiviral pathway (1, 17, 22, 26), there may be other independent mechanisms that warrant a detailed examination of alternative routes that might mediate the recognition of and response to dsRNA, particularly with regard to HCV RNA replication.
ACKNOWLEDGMENTS
We are grateful to Louie Lamorte for generously providing the pISRE reporter construct and for helpful discussions, to Steve Mason for critical reading of the manuscript, and to Michael Cordingley for his support and encouragement of this work. We thank Rongtuan Lin for kindly providing the RANTES luciferase reporter plasmid.
S.A. is a recipient of the Canadian Institutes of Health Research Rx&D postdoctoral research award.
REFERENCES
Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413:732-738.
Ali, S., C. Pellerin, D. Lamarre, and G. Kukolj. 2004. Hepatitis C virus subgenomic replicons in the human embryonic kidney 293 cell line. J. Virol. 78:491-501.
Au, W. C., P. A. Moore, W. Lowther, Y. T. Juang, and P. M. Pitha. 1995. Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc. Natl. Acad. Sci. USA 92:11657-11661.
Blight, K. J., A. A. Kolykhalov, and C. M. Rice. 2000. Efficient initiation of HCV RNA replication in cell culture. Science 290:1972-1974.
Daly, C., and N. C. Reich. 1995. Characterization of specific DNA-binding factors activated by double-stranded RNA as positive regulators of interferon alpha/beta-stimulated genes. J. Biol. Chem. 270:23739-23746.
Doyle, S., S. Vaidya, R. O'Connell, H. Dadgostar, P. Dempsey, T. Wu, G. Rao, R. Sun, M. Haberland, R. Modlin, and G. Cheng. 2002. IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity 17:251-263.
Edelmann, K. H., S. Richardson-Burns, L. Alexopoulou, K. L. Tyler, R. A. Flavell, and M. B. Oldstone. 2004. Does Toll-like receptor 3 play a biological role in virus infections? Virology 322:231-238.
Fitzgerald, K. A., S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S. M. Liao, and T. Maniatis. 2003. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4:491-496.
Foy, E., K. Li, C. Wang, R. Sumpter, Jr., M. Ikeda, S. M. Lemon, and M. Gale, Jr. 2003. Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 300:1145-1148.
Geiss, G., G. Jin, J. Guo, R. Bumgarner, M. G. Katze, and G. C. Sen. 2001. A comprehensive view of regulation of gene expression by double-stranded RNA-mediated cell signaling. J. Biol. Chem. 276:30178-30182.
Genin, P., M. Algarte, P. Roof, R. Lin, and J. Hiscott. 2000. Regulation of RANTES chemokine gene expression requires cooperativity between NF-kappa B and IFN-regulatory factor transcription factors. J. Immunol. 164:5352-5361.
Heylbroeck, C., S. Balachandran, M. J. Servant, C. DeLuca, G. N. Barber, R. Lin, and J. Hiscott. 2000. The IRF-3 transcription factor mediates Sendai virus-induced apoptosis. J. Virol. 74:3781-3792.
Hoebe, K., E. M. Janssen, S. O. Kim, L. Alexopoulou, R. A. Flavell, J. Han, and B. Beutler. 2003. Upregulation of costimulatory molecules induced by lipopolysaccharide and double-stranded RNA occurs by Trif-dependent and Trif-independent pathways. Nat. Immunol. 4:1223-1229.
Kariko, K., H. Ni, J. Capodici, M. Lamphier, and D. Weissman. 2004. mRNA is an endogenous ligand for Toll-like receptor 3. J. Biol. Chem. 279:12542-12550.
Krieger, N., V. Lohmann, and R. Bartenschlager. 2001. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J. Virol. 75:4614-4624.
Lin, R., C. Heylbroeck, P. Genin, P. M. Pitha, and J. Hiscott. 1999. Essential role of interferon regulatory factor 3 in direct activation of RANTES chemokine transcription. Mol. Cell. Biol. 19:959-966.
Lin, R., C. Heylbroeck, P. M. Pitha, and J. Hiscott. 1998. Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation. Mol. Cell. Biol. 18:2986-2996.
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Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, and S. Akira. 2003. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301:640-643.
Yamamoto, M., S. Sato, K. Mori, K. Hoshino, O. Takeuchi, K. Takeda, and S. Akira. 2002. Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J. Immunol. 169:6668-6672.(Samir Ali and George Kuko)
ABSTRACT
The treatment of human embryonic kidney 293 cells harboring a hepatitis C virus (HCV) subgenomic replicon with the double-stranded RNA (dsRNA) mimic poly(I · C) inhibits HCV RNA replication through an undefined mechanism. Interferon regulatory factor 3 (IRF 3) has been widely postulated to mediate various antiviral responses, and its role in mediating the response to dsRNA in 293 cells was examined. Treating the cells with dsRNA did not induce IRF-3 activation, as measured by nuclear localization or the induction of reporter genes. Moreover, the expression of a dominant negative form of IRF-3 did not affect either colony formation upon transfection of subgenomic replicon RNA or the inhibition of the HCV replicon by dsRNA. Our results suggest that the inhibition of HCV RNA replication by poly(I · C) in 293 cells is independent of IRF-3 activation.
TEXT
The establishment of the hepatitis C virus (HCV) subgenomic replicon system in Huh-7 cells (4, 15, 18-20) has permitted a preliminary investigation of the interactions between HCV and host cells. We have established a similar HCV subgenomic replication system in human embryonic kidney (HEK) 293 cells (2, 25). In contrast to what occurs in Huh-7 cells, HCV RNA replication can be inhibited in 293 cells upon treatment with the double-stranded RNA (dsRNA) mimic poly(I · C) (50% inhibitory concentration [IC50], 77 μg/ml].
dsRNA, a by-product of virus replication, is believed to activate the innate immune system and mediate the production of alpha/beta interferon (IFN-/?) and other cytokines (10). dsRNA and poly(I · C) are candidate ligands for Toll-like receptor 3 (TLR-3) (1, 10, 21). Engagement of poly(I · C) with the receptor is proposed to activate a signaling cascade that is MyD88 independent and involves the recruitment of Toll or interleukin 1 receptor domain-containing, adaptor-inducing IFN-? (TRIF) (22, 26, 27), which in turn is postulated to activate various signaling mediators, including IB kinase and TANK-binding kinase 1. These kinases phosphorylate and activate IFN-regulatory factor 3 (IRF-3) (8, 22, 24), which has emerged as a critical mediator of antiviral responses (23). The role of IRF-3 in the induction of antiviral responses by dsRNA remains to be fully elucidated. HEK 293 cells do not normally express TLR-3, and dsRNA can activate IRF-3 in these cells when TLR-3 is ectopically expressed (1, 14). HEK 293 cells harboring the HCV replicon that we had generated are sensitive to dsRNA (2), and they provided a unique tool to examine the role of IRF-3 in mediating the inhibition of HCV RNA replication.
dsRNA does not induce the nuclear localization of IRF-3. The nuclear localization of IRF-3 was examined with a green fluorescent protein (GFP)-IRF-3 construct that fused the GFP coding sequence to the N-terminal portion of IRF-3 in a mammalian expression vector. The GFP-IRF-3 construct was transfected into 293 and Huh-7 cells using FuGene-6 (Roche). The cells were then treated either with 20 hemagglutination units (HAU) of Sendai virus (SenV) per ml (17) or with a dose of two dsRNA mimics, poly(I · C) and poly(U · A), ranging from 50 to 500 μg per ml and then fixed, permeabilized, and stained with DAPI (4',6'-diamidino-2-phenylindole). Transfection of dsRNA into the cells was unnecessary, as adding poly(I · C) to cell culture media strongly inhibits HCV RNA replication in 293 cells (2). In untreated cells, IRF-3 localized primarily to the cytoplasm. As expected, treating S22.3 and LIP3 cells (Huh-7 and 293 cells that harbor the subgenomic replicon, respectively) with SenV and/or poly(I · C) failed to induce the nuclear localization of IRF-3 in either cell line, consistent with the known suppression of IRF-3 activation by the NS3/NS4A protease (9) (Fig. 1A). Treating na?ve 293 and Huh-7 cells with SenV induced clear nuclear localization of IRF-3, suggesting that the activation of IRF-3 is not defective in either cell type, even in Huh-7 cells that do not respond to treatment with dsRNA in inhibiting the replicon RNA (Fig. 1B and C). In contrast to the results of treatment with SenV, na?ve cells treated with 50 to 500 μg of dsRNA per ml showed no effect on IRF-3 localization; extending the treatment with 250 μg of dsRNA per ml for up to 72 h also had no effect on the nuclear localization of IRF-3 [Fig. 1B and C depict representative poly(I · C) concentrations and time points]. The activation of endogenous IRF-3 was also examined using indirect immunofluorescence staining and was found to correlate to the activation pattern of the GFP-IRF-3 reporter. Only SenV, and not poly(I · C), treatment induced the nuclear localization of endogenous IRF-3 (data not shown). Therefore, it appears that dsRNA does not induce the activation of IRF-3 in either na?ve or replicon 293 and Huh-7 cell lines.
Transactivation of the ISRE promoter by IRF-3. The induction of a luciferase reporter gene controlled by IFN-stimulated response elements (ISRE) was used as an alternative method to assess the activation of IRF-3 (3, 5). Cells were transfected with an IRF-3-regulated luciferase reporter containing five ISRE upstream of a firefly luciferase gene (pISRE) and a constitutive Renilla luciferase reporter (pRL-TK; Promega), which served as an internal control. Luciferase readings (Dual-Glo luciferase; Promega) revealed that cells treated with 5 to 50 HAU of SenV per ml induced an approximately 35-fold activation (Fig. 2A); however, no significant activation of the luciferase reporter was observed in cells treated with a dose of dsRNA ranging from 50 to 500 μg/ml. Similarly, a firefly luciferase gene reporter under the control of the IRF-3-regulated RANTES promoter (11, 16) demonstrated that treatment with dsRNA (50 to 500 μg/ml) also did not induce measurable activation relative to the activation induced by SenV treatment (Fig. 2B). Therefore, consistent with the subcellular-localization experiments, dsRNA failed to induce the transactivation of IRF-3-responsive genes, suggesting that IRF-3 is not an activated downstream target of dsRNA in these cells. Na?ve, TLR-3-negative cells were used in the above-described experiments, which may account for the difference in the levels of activation of IRF-3 in response to dsRNA treatment that was previously reported. Either TLR-3-positive cells, ectopic overexpression of TLR-3, or introduction of dsRNA into the intracellular milieu through transfection was employed (1, 6, 14).
Generation of stable cell lines expressing dominant negative IRF-3. Deleting the DNA binding domain of IRF-3 (17) generates a well-characterized dominant negative form, IRF-3 N, which we used to examine the role of IRF-3 in mediating the dsRNA-induced inhibition of HCV RNA replication. Flag-tagged IRF-3 N was constructed and transfected (Lipofectamine 2000; Invitrogen) into 293/FRT cells (Flp-In system; Invitrogen). A stable cell line, 2IN, was generated by selecting cells with 0.2 mg of hygromycin per ml. The stable expression of truncated IRF-3 was confirmed in 2IN cells by using IRF-3- and Flag-specific Western blotting (Fig. 3A). The level of expression of IRF-3 N in these cells was significantly higher than that of the endogenous protein (Fig. 3A), a prerequisite to eliciting the dominant negative effect (compare lanes 293 and 2IN-4).
The dominant negative phenotype of IRF-3 N was confirmed by using the ISRE and RANTES luciferase reporters in na?ve 293, LIP3, and in 2IN cells. Transfecting the three cell lines as described above and treating them with 5 to 50 HAU of SenV per ml revealed that, relative to the induction of luciferase activity in na?ve 293 cells, IRF-3 activation was suppressed in LIP3 replicon cells due to the presence of the NS3/NS4A protease (9) and was diminished in 2IN cells due to the expression of IRF-3 N (Fig. 3B). This result confirms that the dominant negative form interferes with the function of endogenous IRF-3, as previously reported (12, 17). Basal luciferase activities were comparable in luciferase reporter-transfected cells expressing IRF-3 N (2,776 ± 95 cps) and wild-type IRF-3 (1,895 ± 274 cps), indicating that the transfection procedure did not induce the activation of IRF-3.
Dominant negative IRF-3 does not affect HCV RNA replication. Transfection into na?ve 293 cells of total RNA obtained from 293 cells harboring the replicon generates approximately 7.5 x 103 neomycin-resistant colonies per μg of replicon RNA (2). In order to assess the effect of the stable expression of dominant negative IRF-3 on the efficiency of colony formation, 20 μg of total RNA isolated from 293Rep17 cells containing an estimated 107 replicon copies per μg was transfected as described previously (2) into 2PD cells (equivalent to na?ve 293 cells) (stable cells were generated by transfecting the empty Flp-In vector), 2IW cells (293 cells stably overexpressing wild-type IRF-3), or 2IN cells. Selecting the cells with 0.6 μg of neomycin per ml, staining the cells with crystal violet, and quantifying the resulting colonies indicated that there was no measurable effect on the number of replicon colonies generated with 2IN cells compared to the number of 293 or 2IW cells in repeated experiments (Fig. 4A). Therefore, IRF-3 does not appear to mediate an antiviral response expected to be activated by the presence of replicon RNA in the cells.
Several replicon-containing 2IN cells were expanded into cell lines, termed 2NR cells, and the maintenance of the IRF-3 N phenotype in these expanded cell lines was confirmed (data not shown).
IRF-3 does not modulate HCV RNA inhibition by dsRNA. dsRNA potently inhibits HCV RNA replication in 293 cells harboring the replicon (IC50, 77 μg/ml) (2). Treating na?ve 293, LIP3, and 2NR cells (replicon 293 cells stably expressing IRF-3 N) with a 3.9- to 1,000-μg/ml dose of poly(I · C) for 72 h revealed that the stable overexpression of dominant negative IRF-3 N did not desensitize the cells and shift the IC50 of dsRNA. The levels of inhibition of HCV RNA replication were comparable in LIP3 and 2NR cells, yielding IC50s of 63.5 ± 8.9 μg/ml for LIP3 cells and 44.7 ± 3.1 μg/ml for 2NR cells, as measured by TaqMan real-time reverse transcription-PCR (2) (Fig. 4 B). Therefore, IRF-3 does not appear to mediate an essential role in the inhibition of HCV replicons by dsRNA.
Our combined data examining IRF-3 subcellular localization, transactivation, and dominant negative suppression indicate that IRF-3 is not activated by topical dsRNA treatment and that the inhibitory response to dsRNA on HCV RNA replication observed in 293 cells is IRF-3 independent.
A recent report by Edelmann et al. (7) questioned the role of TLR-3 as the sole recognition element mediating antiviral responses to dsRNA, and a study by Hoebe et al. (13) reported TLR-3/TRIF axis-independent antiviral responses to dsRNA. Therefore, it appears that in addition to the reported TLR-3/TRIF/IRF-3 antiviral pathway (1, 17, 22, 26), there may be other independent mechanisms that warrant a detailed examination of alternative routes that might mediate the recognition of and response to dsRNA, particularly with regard to HCV RNA replication.
ACKNOWLEDGMENTS
We are grateful to Louie Lamorte for generously providing the pISRE reporter construct and for helpful discussions, to Steve Mason for critical reading of the manuscript, and to Michael Cordingley for his support and encouragement of this work. We thank Rongtuan Lin for kindly providing the RANTES luciferase reporter plasmid.
S.A. is a recipient of the Canadian Institutes of Health Research Rx&D postdoctoral research award.
REFERENCES
Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413:732-738.
Ali, S., C. Pellerin, D. Lamarre, and G. Kukolj. 2004. Hepatitis C virus subgenomic replicons in the human embryonic kidney 293 cell line. J. Virol. 78:491-501.
Au, W. C., P. A. Moore, W. Lowther, Y. T. Juang, and P. M. Pitha. 1995. Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc. Natl. Acad. Sci. USA 92:11657-11661.
Blight, K. J., A. A. Kolykhalov, and C. M. Rice. 2000. Efficient initiation of HCV RNA replication in cell culture. Science 290:1972-1974.
Daly, C., and N. C. Reich. 1995. Characterization of specific DNA-binding factors activated by double-stranded RNA as positive regulators of interferon alpha/beta-stimulated genes. J. Biol. Chem. 270:23739-23746.
Doyle, S., S. Vaidya, R. O'Connell, H. Dadgostar, P. Dempsey, T. Wu, G. Rao, R. Sun, M. Haberland, R. Modlin, and G. Cheng. 2002. IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity 17:251-263.
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