Characterization of the interferon regulatory factor 3-mediated antiviral response in a cell line deficient for IFN production

Molecular Immunology 46 (2009) 393–399

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Characterization of the interferon regulatory factor 3-mediated antiviral response in a cell line deficient for IFN production Tracy Chew a , Ryan Noyce b , Susan E. Collins a , Meaghan H. Hancock a,1 , Karen L. Mossman a,b,∗ a b

Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, L8N 3Z5 Canada Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, L8N 3Z5 Canada

a r t i c l e

i n f o

Article history: Received 4 October 2008 Accepted 14 October 2008 Available online 26 November 2008 Keywords: IRF-3 Vero cells IFN Virus Innate immunity

a b s t r a c t The innate cellular response to virus particle entry in non-immune cells requires the transcriptional activity of interferon regulatory factor 3 (IRF-3), but not production of type I interferon (IFN). Here, we characterize the IFN-independent innate cellular response to virus-derived stimuli in Vero cells, a monkey kidney epithelial cell line deficient for IFN production. We provide evidence that Vero cells are deficient in their ability to mount an IRF-3-dependent, IFN-independent antiviral response against either incoming virus particles or polyinosinic:polycytidylic acid (pIC), a dsRNA mimetic. We further demonstrate that abundance of IRF-3 protein is a determinant in the pIC-mediated antiviral signalling pathway. These observations further characterize the permissive nature of Vero cells to viral infection, and highlight the crucial involvement of IRF-3 in the innate antiviral response. © 2008 Elsevier Ltd. All rights reserved.

1. Introduction The innate cellular response to viral infection involves the expression of multiple antiviral genes, the most potent of which are type I interferons (IFNs). Type I IFNs, comprised mostly of IFN␣ and ␤, are a family of pleiotropic cytokines whose expression is largely dependent on the transcription factor IFN regulatory factor (IRF)-3 (Au et al., 1995; Juang et al., 1998; Sato et al., 2000). Through production of IFN, viruses induce the expression of a large number of antiviral factors known as IFN-stimulated genes (ISGs) (de Veer et al., 2001), which play distinct, yet overlapping, roles in inducing an antiviral response in cells. These roles include inhibition of host and viral protein translation (Guo et al., 2000b; Hui et al., 2003), recruitment of NK cells to sites of infection (D’Cunha et al., 1996), regulation of cellular proliferation and apoptosis (Marques et al., 2005), and regulation of adaptive immunity (Escors et al., 2008; Le Bon et al., 2003). Together, these antiviral effectors are sufficient to inhibit further viral gene expression and virus replication. The mechanisms by which different viruses induce an IFNmediated antiviral response are distinct but appear to converge at the activation of Tank binding kinase 1 (TBK-1) and Inhibitor of

∗ Corresponding author at: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, L8N 3Z5 Canada. Fax: +1 905 522 6750. E-mail address: [email protected] (K.L. Mossman). 1 Current address: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada. 0161-5890/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2008.10.010

NF␬B kinase epsilon (IKK␧), the kinases that phosphorylate IRF3 (Kato et al., 2006; Sasai et al., 2006; Yoneyama et al., 2004). Phosphorylation of IRF-3 induces a conformational change that exposes its IRF association domain (IAD), leading to homodimerization and subsequent nuclear translocation (Kumar et al., 2000; Lin et al., 1999). Here, activated IRF-3 binds to the promoter region of the IFN␤ gene and, along with other transcription factors such as activator protein (AP)-1 and CREB binding protein (CBP)/p300, leads to expression and secretion of IFN␤ (Escalante et al., 2007; Qin et al., 2005; Wathelet et al., 1998). IFN␤ signals autocrinely and paracrinely via JAK/STAT signalling and ultimately leads to the expression of the full subset of IFN␣ genes and ISGs including IRF-7, ISG56, and ISG15 (de Veer et al., 2001). The cellular recognition of viral components appears to depend both on the structure and subcellular location of the pathogen-associated molecular pattern (PAMP). For example, Toll-like receptor (TLR) 3 has been shown to recognize polyinosinic:polycytidylic acid (pIC), a synthetic mimic of dsRNA, presumably in the endosomal compartment (Alexopoulou et al., 2001). Conversely, intracellular dsRNA has been shown to be recognized by the cytosolic RNA helicases melanoma differentiation associated gene (MDA) 5 and retinoic acid inducible gene (RIG)-I in a length-dependent manner (Kato et al., 2006, 2008). Furthermore, the structural differences between self- and non-self-dsRNA determine whether an immune response is mounted (Marques et al., 2006). Thus, it is clear that distinct but overlapping pathways govern IRF-3 activation in response to different stimuli, and that the regulation of these pathways is likely diverse as well.

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Our lab and others have shown that the induction of an antiviral state does not require virus replication and can occur in the absence of IFN signalling (Boyle et al., 1999; Mossman et al., 2001; Netterwald et al., 2004; Prescott et al., 2005; Preston et al., 2001; tenOever et al., 2002). We have previously demonstrated that the cellular response to virus particle entry requires IRF-3 and induces ISGs in the absence of IFN production and viral gene expression in primary fibroblasts (Collins et al., 2004; Paladino et al., 2006). Although the TLR and RIG-I pathways are not essential for this response to occur (Paladino et al., 2006), additional viral and/or cellular components that are required for this response remain largely unknown. The establishment of an IFN-independent antiviral state involves the production of a subset of ISGs whose promoters interact directly with IRF-3 via the interferon-stimulated response element (ISRE). These genes include ISG56, which has been shown to inhibit protein translation by interacting with eukaryotic initiation factor 3 (eIF-3) (Guo et al., 2000a), and ISG15, which is involved in NK cell activation and regulation of other antiviral proteins (D’Cunha et al., 1996; Kim et al., 2008). Importantly, the IRF-3-dependent, IFN-independent response to virus particle entry occurs independently of any detectable soluble factor, since supernatants from treated cells were unable to confer protection to naïve cells (Collins et al., 2004; Paladino et al., 2006). In addition, virus particle-induced transcription of ISGs has been shown to occur in the presence of cycloheximide, demonstrating that production of IFN and subsequent signalling is not required for this response (Nicholl et al., 2000; Zhu et al., 1997). Therefore, this process is likely to be an intracellular host response, occurring independently of secreted cytokines such as the IFN proteins. To further characterize the IFN-independent cellular response to virus particle entry, we initiated studies in a cell type deficient for IFN production. Vero cells, derived from African Green monkey kidney epithelia, contain a genetic lesion in the IFN␤ locus (Emeny and Morgan, 1979; Mosca and Pitha, 1986; Wathelet et al., 1992). These cells are extremely permissive to viral infection because of their inability to produce IFN, and are widely used to propagate virus and vaccine stocks, perform IFN bioassays and study virus–host interactions. We demonstrate here that Vero cells, in addition to their inability to produce IFN␤, are also defective in their ability to produce ISGs and mount an antiviral state in an IFN-independent manner. We further show that the nature of this defective IFN-independent antiviral response can be attributed to low endogenous expression of IRF-3. Finally, we show that pICinduced antiviral signalling is absent in these cells and can be restored upon exogenous expression of IRF-3.

lacks the envelope glycoprotein G required for packaging and egress (Lai et al., 2008). Replication-deficient virus particles were prepared by treating viruses with ultraviolet light at an energy that abrogated viral protein expression as monitored by immunofluorescence (Collins et al., 2004). Cells were infected in serum-free medium for 1 h at 37 ◦ C, with the exception of adenoviruses, where infection occurred in PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2 for 30 min at room temperature.

2. Materials and methods

Protein from mock-infected or treated cells was harvested 24 h post-treatment in a whole cell lysis buffer containing 20 mM HEPES pH 7.4, 100 mM NaCl, 10 mM b-glycerophosphate, 50 mM NaF, 1 mM Na3 VO4 , 1 mM phenylmethyl sulfonyl fluoride, 2 mM dithiothreitol, and 1× protease inhibitor cocktail (Sigma). Cells were lysed for 10 min on ice, then centrifuged at 13 000 × g for 10 min and supernatants collected and boiled in sample buffer containing SDS and ␤-mercaptoethanol. Samples were quantified by Bradford assay kit (Bio-Rad) and subject to immunoblot analysis for IRF3. Blots were subsequently probed for actin as a loading control. Quantitative immunoblots were performed using ECL AdvanceTM Western blotting detection kit (GE Healthcare) as directed. Chemifluorescent signal was scanned and quantified on a TyphoonTM Variable Mode Imager (GE Healthcare) as above. Relative fluorescent intensity was expressed as a percentage of HEL or Vero IRF-3 protein and normalized to actin loading control. Statistical analysis was performed using one-way ANOVA or Student’s t-test as indicated.

2.1. Cells, viruses, and infections Vero cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 5% fetal bovine serum, l-glutamine, and antibiotics (5% DMEM). Human lung fibroblasts (HEL) and osteosarcoma cells (U2OS) were maintained in 10% DMEM. A549 lung epithelial cells were maintained in 10% ␣MEM. All cell lines were obtained from American Type Culture Collection. AdhuIRF3, a replication-deficient adenovirus encoding human IRF-3, was a kind gift from Dr. Jonathan Bramson (McMaster University, Hamilton, Ontario, Canada). AdBHGE1E3 (AdE1E3) is the parental control (Ng and Graham, 2002). Cells were infected at a multiplicity of infection (moi) of 10 pfu/cell for AdE1E3, AdhuIRF3, and Herpes simplex virus 1 (HSV-1, KOS strain), and at a moi of 20 HAU/106 cells for Sendai Virus (SeV, Cantell strain). VSV-GFP has been previously described (Stojdl et al., 2003). VSV-GFP(G)

2.2. Antiviral state assays Cells were mock treated or infected with AdhuIRF-3 or AdE1E3 for 24 h, then treated with replicating or UV-inactivated virus, 100 units huIFN␣ (100 U/mL, Sigma), or polyinosinic-polycytidylic acid (pIC) (100 ug/mL, Amersham-Pharmacia). Treated cells were then challenged 24 h later with VSV-GFP and total fluorescence monitored at 24 h post-challenge (hpc) using a TyphoonTM Variable Mode Imager (GE Healthcare). Relative fluorescence was quantified using ImageQuantTM TL software (GE Healthcare). Alternatively, cells were co-transfected with 1 ug test plasmid and 1 ug pSG5-VSV-G (Stratagene) as described (Lai et al., 2008). Briefly, cotransfected cells were treated as indicated for 24 h then challenged with VSV-GFP(G). Supernatants from challenged monolayers were collected at 24 hpc and titered on Vero cells, using GFP fluorescence to assess viral gene expression. Relative fluorescence was quantified as a percentage of mock treated samples, and significance performed by one-way ANOVA with post hoc tests as indicated. 2.3. RT-PCR RNA from mock-infected or treated cells was harvested 8 h posttreatment in Trizol reagent (Invitrogen) as directed. 1–2 ug RNA were reverse transcribed using random hexamer primers and SSII reverse transcriptase (Invitrogen) and amplified using Taq polymerase (Invitrogen) as recommended using primers for human ISG56 or GAPDH as previously described (Collins et al., 2004). 2.4. Transfections Transfections were performed in six-well plates using Lipofectamine 2000 (Invitrogen) as directed. Cells were harvested or treated 24 h later. 2.5. Immunoblotting

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2.6. IRF-3 cloning and sequence analysis IRF-3 mRNA was reverse transcribed and PCR amplified as described using Platinum Taq HIFI polymerase (Invitrogen) with primers flanking wild-type human IRF-3. Purified PCR products were cloned into sequencing vector pCST, a kind gift from Duncan Chong (McMaster University, Hamilton, Ontario, Canada), or pBluescriptII KS (+/−) (Stratagene). A minimum of two independently derived PCR products were sequenced on both strands to ensure the accuracy of the sequence. IRF-3 sequences were then subcloned into pCMVTnT (Promega) for mammalian expression. 3. Results 3.1. Vero cells fail to mount an IFN-independent antiviral response Vero cells are kidney epithelial cells isolated from a normal African green monkey that can respond to, but not produce, IFN␤ (Emeny and Morgan, 1979; Mosca and Pitha, 1986; Wathelet et al., 1992). They are widely used to propagate virus and to investigate virus-mediated cellular signalling events. As antiviral activity observed in these cells is independent of de novo IFN␤ synthesis, we assessed the ability of Vero cells to undergo an antiviral state in response to various viral stimuli. Vero cells were treated for 24 h with huIFN␣, pIC or UV-inactivated Herpes simplex virus type 1 (HSV-1, a representative DNA virus) or Sendai virus (SeV, a representative RNA virus) and then challenged with VSV-GFP. Whereas exogenous IFN treatment resulted in >75% reduction in GFP fluorescence, neither pIC nor virus particle treatment significantly affected GFP fluorescence (Fig. 1). These results demonstrate that, in contrast to primary human fibroblast cells (Collins et al., 2004), Vero cells fail to mount an IFN-independent antiviral response. 3.2. Expression of human IRF-3 restores antiviral immunity against pIC but not UV-inactivated virus The IFN-independent induction of antiviral genes is thought to rely on IRF-3 activity (Collins et al., 2004; Grandvaux et al., 2002; Peters et al., 2002). To understand the contribution of IRF-3 to antiviral signalling in Vero cells, we expressed human IRF-

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3 using an adenoviral vector (AdhuIRF-3). In control adenovirus treated cells, reproducible accumulation of ISG56 mRNA was only observed following treatment with IFN or infection with replication competent SeV (Fig. 2a). Pretreatment with AdhuIRF-3 additionally stimulated ISG56 mRNA accumulation in response to pIC and enhanced the response to SeV. Similar results in ISG enhancement were observed with ISG15 protein expression as monitored by immunofluorescence microscopy (data not shown). Consistent with this observation, AdhuIRF-3 pretreatment led to restoration of pIC-induced antiviral immunity in a standard antiviral assay (Fig. 2b and c). Supernatants transferred from treated cells were unable to confer antiviral protection to naïve Vero cells (Fig. 2d), indicating that production of soluble factors do not play a role in the generation of an innate antiviral response. Collectively, these results suggest that expression of human IRF-3 restores the pIC-mediated antiviral response in Vero cells. 3.3. Sequence analysis of IRF-3 cloned from Vero cells Since exogenous huIRF-3 expression restored selective antiviral functions in Vero cells, we cloned Vero IRF-3 to investigate sequence differences. Vero IRF-3 is 96% identical to human IRF-3 at the amino acid level and contains two deletion sites corresponding to aa164–168 and aa301 of human IRF-3 (Fig. 3). These deletion sites are situated within the transactivation domain (TAD) and the IAD, respectively. As these sites are important for dimerization and nuclear translocation of IRF-3 following stimulation, it is possible that these differences play a role in the ability of endogenous IRF-3 to induce antiviral gene expression in Vero cells. 3.4. Endogenous Vero IRF-3 demonstrates altered mobility and abundance relative to human cells To further characterize differences between the two species of IRF-3, immunoblot analysis was performed. While the ∼55 kDa IRF3 doublet was observed in varying abundance between human cell lines, IRF-3 from Vero cells displayed a doublet with an apparent faster migration and at a significantly lower level of expression (Fig. 4a and b). The altered migration of Vero IRF-3 could be explained by the five amino acid difference in protein length, differences in post-translational modification of individual IRF-3 residues that differ between the two species, or to differences in the post-translational modification machinery in human and monkey cells. Regardless of the mechanism, this observation suggests that IRF-3 in Vero cells has altered mobility and is expressed at significantly lower levels than in human cells that have an intact IRF-3-dependent signalling pathway. A similar phenotype was observed in CV-1 African green monkey fibroblast cells, with respect to IRF-3 migration and abundance, and response to pIC (Supplemental data). 3.5. Abundance of IRF-3 in Vero cells is a determinant of pIC-induced antiviral immunity

Fig. 1. Vero cells fail to mount an IFN-independent antiviral response. (a) Cells were treated for 24 h with IFN, pIC, HSV-1 UV, or SeV UV then challenged with VSV-GFP. Monolayers were imaged 24 h post-challenge for GFP fluorescence, indicative of viral replication. (b) Quantification of GFP fluorescence from (a). Error bars represent S.E.M. from three independent experiments. (*) Represents statistical significant by one-way ANOVA using Tukey’s post hoc test (p < 0.001).

Given that endogenous IRF-3 in Vero cells is expressed at lower levels relative to IRF-3 in human cells, and demonstrates differences in virus-mediated activation relative to human IRF-3, we investigated whether differences in protein abundance could explain the lack of antiviral activity. Cells were co-transfected with a parental vector, human IRF-3 or Vero IRF-3, along with a plasmid encoding the envelope glycoprotein G from VSV. Transfected cells were treated as indicated and then challenged with VSV-GFP(G), which can only replicate in cells transfected with the G expression vector, thus permitting the quantification of antiviral activity of various IRF-3 constructs independent of transfection efficiency (Lai et al.,

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Fig. 2. Human IRF-3 expression restores antiviral immunity against pIC. Cells were preloaded for 24 h with AdhuIRF-3 or its control vector, and then treated as indicated. (a) RNA was harvest 8 h post-treatment and subject to RT-PCR for ISG56 or GAPDH as indicated. (b) Cells were challenged 24 h post-treatment with VSV-GFP and monolayers scanned for GFP fluorescence as a measure of viral gene expression. (c) Quantification of relative GFP fluorescence from (b). Error bars represent S.E.M. from three independent experiments. Statistical significance by one-way ANOVA and Tukey’s post hoc test is represented by (*) (p < 0.05) and (**) (p < 0.001). (d) Supernatants from cells treated in (b) were transferred to naïve Vero cells for 24 h, then challenged with VSV-GFP and monolayers scanned for GFP fluorescence. (e) Protein samples from mock- or Ad-pretreated cells were subjected to Western blot analysis for IRF-3. For visualization purposes, 40 ug of whole cell extract from mock and AdE1E3 treated cells were loaded along with 10 ug of whole cell extract from AdhuIRF-3 treated cells.

2008). Virus contained within supernatants from challenged cells was subsequently titered on naive Vero cells, and relative GFP expression was quantified as a measure of virus production. Relative expression of IRF-3 from mock and transfected cells is depicted in Fig. 5a and b. As observed with AdhuIRF-3 treatment, transfected human IRF-3 restored pIC-induced antiviral immunity that approached the level of IFN-induced protection (Fig. 5c). Furthermore, transfection of Vero IRF-3 also restored responsiveness to pIC. Interestingly, no difference in antiviral activity against virus parti-

cles was detected in cells transfected with either control plasmid or plasmid expressing human or Vero IRF-3. These data imply that the endogenous IRF-3 expression level in Vero cells plays a dominant role in determining antiviral activity against pIC. Furthermore, since the difference in mobility between human IRF-3 and Vero IRF-3 is maintained following IRF-3 expression in Vero cells (Fig. 5a), differences in the post-translational modification machinery in human cells versus monkey cells likely does not account for the altered mobility.

Fig. 3. Amino acid sequence of IRF-3 in Vero cells versus human cells. Differences and deletions are represented in bold and highlighting. Functional domains are indicated. DBD, DNA binding domain; NES, nuclear export signal; TAD, transactivation domain; IAD, IRF association domain; SRR, serine-rich region.

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Fig. 4. IRF-3 in Vero cells has lower abundance and faster migratory pattern on SDS-PAGE. (a) 15 ug of whole cell lysate was subjected to Western blot analysis from four different cell lines: HEL primary lung fibroblasts, A549 lung epithelia, U2OS osteosarcoma, and Vero monkey kidney epithelia. Blots were first probed for IRF3 and visualized by enhanced chemifluorescence, then subsequently reprobed for actin. Relative chemifluorescence was normalized to actin and is depicted in (b). (*) Represents statistical significance by one-way ANOVA and Newman–Keul’s post hoc test (p < 0.05).

4. Discussion While the pathways involved in IFN production and signalling following virus infection have been relatively well characterized, few components aside from TBK-1 and IRF-3 have been identified for the IFN-independent intracellular antiviral response. The biological significance of this response is likely realized under conditions of low multiplicity infection or in response to defective virus particles, and is predicted to be the predominant response in the first cells to be infected within a given host (Paladino et al., 2006). Vero cells are widely used in the study of virus–host

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interactions and are commonly used for growing virus and vaccine stocks. Furthermore, viruses that normally demonstrate a very narrow host range, due to their evolutionary history within a single species, are often propagated and/or titered in Vero cells or related African green monkey kidney cells (e.g. myxoma virus (Best et al., 2000; Mossman et al., 1995)). Here we provide evidence that Vero cells have a dysfunctional intracellular antiviral signalling pathway over and above their inability to produce IFN␤, which likely explains the permissive nature of Vero cells to viral infection. We also demonstrate that the endogenous abundance of IRF-3 protein in Vero cells is a key determinant in their ability to respond to pIC stimulation. The IFN-independent response does not rely on prototypic antiviral signalling pathways. It has been demonstrated that p38 MAPK, JNK/SAPK, and ERK 1/2 are not essential (Noyce et al., 2006). TLR as well as RIG-I signalling were likewise shown to be nonessential despite their classical involvement in IRF-3 activation following PAMP recognition (Paladino et al., 2006). A PI3K-related family member is involved in the response; however, identification of the kinase(s) remains to be determined (Noyce et al., 2006). Soluble virus-encoded glycoproteins that mediate cellular binding and entry have been implicated in eliciting the IFN-independent antiviral response (Boehme et al., 2004; Hidmark et al., 2005). However, the virus recognition event is currently unclear. Thus, the signalling pathway that governs IRF-3 activation in response to virus particle entry is of interest as it represents the first line of defense against incoming pathogens. We observed that AdhuIRF-3 pretreatment restored pICinduced ISG56 mRNA accumulation and antiviral activity in a VSV challenge model. Interestingly, AdE1E3 infection reduced VSVGFP fluorescence in response to pIC to ∼70% of mock, suggesting that adenovirus infection may sensitize cells to pIC. However, full sensitization to pIC required AdhuIRF-3 pretreatment, demonstrating a significant involvement of IRF-3 in the antiviral response. This protection is intracellular, as supernatants failed to confer antiviral protection on naïve cells. Thus, in addition to the failure to produce IFN␤, Vero cells fail to induce soluble antiviral cytokines in response to virus particle treatment. The lack of IFN␣ production may indicate a requirement for IFN␤ production, as suggested

Fig. 5. Human and Vero IRF-3 overexpression restore the ability of Vero cells to inhibit viral replication in response to pIC in a transfection system. (a) Whole cell lysates from Vero cells transfected with either human or Vero IRF-3 were prepared and 5 ug lysate were subjected to Western blot analysis as in Fig. 4 and quantified (b). (c) Cells were co-transfected with a control vector, human IRF-3, or Vero IRF-3 along with pSG5-VSV-G. Transfected cells were treated as indicated and challenged 24 h later with VSVGFP-G. Twenty four hours post-challenge, supernatants were collected and titered on naive Vero cells, monitoring GFP fluorescence as a measure of relative viral titers. Error bars represent S.E.M. from three independent experiments. (**) Represents significance by one-way ANOVA and Tukey’s post hoc test compared to the mock sample in each treatment group (p < 0.001).

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for fibroblast cells (Erlandsson et al., 1998), thus demonstrating a further impairment in the antiviral response in this cell line. Importantly, the inability of supernatants to confer antiviral protection to naïve cells implies a lack of production of any effective antiviral cytokine, which may include not only the type I IFNs, but also other species of IFN such as IFN␭, which has been shown to be an important antiviral cytokine in epithelial cells (Contoli et al., 2006; Sommereyns et al., 2008). The ability of SeV but not pIC, a dsRNA mimetic, to produce ISGs in Vero cells was initially unexpected. One explanation for this observation may be the level of stimulation between the two treatments. SeV replication involves multiple rounds of amplification of its genome and hence of viral dsRNA production. However, treatment of Vero cells with up to 400 ug/ml pIC failed to induce ISG56 mRNA or an antiviral state (data not shown). While pIC is utilized as a dsRNA mimic, distinct mechanisms of IRF-3 activation have been demonstrated between SeV and dsRNA, specifically with respect to the involvement of TLR3 and the RNA helicases (Elco et al., 2005; Kato et al., 2006), which likely explains the differential activation in Vero cells to these two stimuli. The inability of Vero cells to mount an IFN-independent antiviral response against virus particles was surprising, but given the uncharacterized nature of this pathway, the explanation for this observation is less clear. These data do, however, confirm our previous observations that the IFN-independent antiviral response involves a novel signal transduction pathway that converges on TBK-1 and IRF-3 (Noyce et al., 2006). As expected, we failed to observe induction of ISGs in response to replication competent HSV-1, as mechanisms of blocking antiviral gene expression have been described. For example, ICP0 of HSV-1 has been shown to block IFN and ISG production via IRF-3 and IRF-7 (Lin et al., 2004; Mossman and Smiley, 2002). In addition, the ability of HSV-1 to affect global gene expression was reflected in changes in relative GAPDH signal, and has been previously observed late times post-infection (Collins et al., 2004). We have observed differences in the sequence, mobility, and abundance of endogenous IRF-3 between Vero cells and human cells. The mobility and abundance differences are likely attributed to species-specific differences, since IRF-3 from CV-1 cells demonstrated similar mobility and abundance to IRF-3 from Vero cells on SDS-PAGE, and expression of exogenous human IRF-3 in Vero cells retained its slower migration pattern relative to expression of exogenous Vero IRF-3, despite similar expression levels. While we have demonstrated that IRF-3 abundance is a major determinant to pIC-induced antiviral signalling in the absence of IFN, we cannot rule out the possibility that sequence differences or deletions contribute, in part, to the function and activation of IRF-3 and subsequent signalling. To our knowledge, this is the first example of a non-mutagenized cell type deficient for innate antiviral signalling due to low endogenous levels of IRF-3. While the exogenous expression of either human of Vero IRF3 restored pIC-induced antiviral gene expression and protection from subsequent viral challenge, suggesting that the endogenous abundance of IRF-3 is a major determinant in the ability of Vero cells to respond to pIC, these cells remained unresponsive to virus particle entry, highlighting the distinct nature of IRF-3 activation in response to different viral stimuli. The selective restoration of pIC-induced antiviral immunity suggests that in response to virus particle entry, one or multiple cellular components that underlie entry-mediated IRF-3 activation are dysfunctional, and that this component(s) is not required for dsRNA signalling. Alternatively, Vero cells may express a cellular component that selectively inhibits IRF-3 activity in response to virus entry, although this is less likely given that cells tend to harbor loss-of-function rather than gain-of-function mutations.

5. Conclusion In summary, we have demonstrated that Vero cells have defective pIC-induced antiviral immunity that can be attributed to low endogenous levels of IRF-3 protein. This observation is particularly relevant to virologists due to the widespread use of this cell line in the study of antiviral immunity. In addition, these observations further explain the permissive nature of Vero cells to various viruses, making them an ideal candidate for the propagation of high titer virus and vaccine stocks. Importantly, experiments using this cell line should be interpreted with caution, as the antiviral signalling pathway in these cells is atypical. Acknowledgments The authors thank D.T. Cummings for technical assistance, J.L. Hummel and P. Paladino for helpful discussions, and Drs. J.L. Bramson and B.D. Lichty for reagents, cells, and viruses. This work was supported by an operating grant from the Canadian Institutes of Health Research. KLM is the recipient of an Rx&D/CIHR Career Award. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molimm.2008.10.010. References Alexopoulou, L., Holt, A.C., Medzhitov, R., Flavell, R.A., 2001. Recognition of doublestranded RNA and activation of NF-[kappa]B by Toll-like receptor 3. Nature 413, 732–738. Au, W., Moore, P., Lowther, W., Juang, Y., Pitha, P., 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. PNAS 92, 11657–11661. Best, S.M., Collins, S.V., Kerr, P.J., 2000. Coevolution of host and virus: cellular localization of virus in myxoma virus infection of resistant and susceptible European rabbits. Virology 277, 76–91. Boehme, K.W., Singh, J., Perry, S.T., Compton, T., 2004. Human cytomegalovirus elicits a coordinated cellular antiviral response via envelope glycoprotein B. J. Virol. 78, 1202–1211. Boyle, K.A., Pietropaolo, R.L., Compton, T., 1999. Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol. Cell. Biol. 19, 3607–3613. Collins, S.E., Noyce, R.S., Mossman, K.L., 2004. Innate cellular response to virus particle entry requires IRF3 but not virus replication. J. Virol. 78, 1706–1717. Contoli, M., Message, S.D., Laza-Stanca, V., Edwards, M.R., Wark, P.A., Bartlett, N.W., Kebadze, T., Mallia, P., Stanciu, L.A., Parker, H.L., Slater, L., Lewis-Antes, A., Kon, O.M., Holgate, S.T., Davies, D.E., Kotenko, S.V., Papi, A., Johnston, S.L., 2006. Role of deficient type III interferon-lambda production in asthma exacerbations. Nat. Med. 12, 1023–1026. D’Cunha, J., Knight, E., Haas, A.L., Truitt, R.L., Borden, E.C., 1996. Immunoregulatory properties of ISG15, an interferon-induced cytokine. PNAS 93, 211–215. de Veer, M.J., Holko, M., Frevel, M., Walker, E., Der, S., Paranjape, J.M., Silverman, R.H., Williams, B.R.G., 2001. Functional classification of interferon-stimulated genes identified using microarrays. J. Leukoc. Biol. 69, 912–920. Elco, C.P., Guenther, J.M., Williams, B.R., Sen, G.C., 2005. Analysis of genes induced by Sendai virus infection of mutant cell lines reveals essential roles of interferon regulatory factor 3, NF-kappaB, and interferon but not toll-like receptor 3. J. Virol. 79, 3920–3929. Emeny, J.M., Morgan, M.J., 1979. Susceptibility of various cells treated with interferon to the toxic effect of poly(rI).poly(rC) treatment. J. Gen. Virol. 43, 253–255. Erlandsson, L., Blumenthal, R., Eloranta, M.L., Engel, H., Alm, G., Weiss, S., Leanderson, T., 1998. Interferon-beta is required for interferon-alpha production in mouse fibroblasts. Curr. Biol. 8, 223–226. Escalante, C., Nistalvillan, E., Shen, L., Garciasastre, A., Aggarwal, A., 2007. Structure of IRF-3 bound to the PRDIII-I regulatory element of the human interferon-␤ enhancer. Molecular Cell 26, 703–716. Escors, D., Lopes, L., Lin, R., Hiscott, J., Akira, S., Davis, R.J., Collins, M.K., 2008. Targeting dendritic cell signalling to regulate the response to immunisation. Blood 10.1182, 2408. Grandvaux, N., Servant, M., tenOever, B., Sen, G.C., Balachandran, S., Barber, G.N., Lin, R., Hiscott, J., 2002. Transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes. J. Virol. 76, 5532–5539.

T. Chew et al. / Molecular Immunology 46 (2009) 393–399 Guo, J., Hui, D.J., Merrick, W.C., Sen, G.C., 2000a. A new pathway of translational regulation mediated by eukaryotic initiation factor 3. EMBO J. 19, 6891–6899. Guo, J., Peters, K.L., Sen, G.C., 2000b. Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection. Virology 267, 209–219. Hidmark, A.S., McInerney, G.M., Nordstrom, E.K., Douagi, I., Werner, K.M., Liljestrom, P., Karlsson Hedestam, G.B., 2005. Early alpha/beta interferon production by myeloid dendritic cells in response to UV-inactivated virus requires viral entry and interferon regulatory factor 3 but not MyD88. J. Virol. 79, 10376–10385. Hui, D.J., Bhasker, C.R., Merrick, W.C., Sen, G.C., 2003. Viral stress-inducible protein p56 inhibits translation by blocking the interaction of eIF3 with the ternary complex eIF2·GTP·Met-tRNAi. J. Biol. Chem. 278, 39477–39482. Juang, Y., Lowther, W., Kellum, M., Au, W., Lin, R., Hiscott, J., Pitha, P.M., 1998. Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3. PNAS 95, 9837–9842. Kato, H., Takeuchi, O., Sato, S., Yoneyama, M., Yamamoto, M., Matsui, K., Uematsu, S., Jung, A., Kawai, T., Ishii, K.J., Yamaguchi, O., Otsu, K., Tsujimura, T., Koh, C., Reis e Sousa, C., Matsuura, Y., Fujita, T., Akira, S., 2006. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441, 101–105. Kato, H., Takeuchi, O., Mikamo-Satoh, E., Hirai, R., Kawai, T., Matsushita, K., Hiiragi, A., Dermody, T.S., Fujita, T., Akira, S., 2008. Length-dependent recognition of doublestranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5. J Exp Med 205, 1601–1610. Kim, M.J., Hwang, S.Y., Imaizumi, T., Yoo, J.Y., 2008. Negative feedback regulation of RIG-I-mediated antiviral signaling by interferon-induced ISG15 conjugation. J. Virol. 82, 1474–1483. Kumar, K.P., McBride, K.M., Weaver, B.K., Dingwall, C., Reich, N.C., 2000. Regulated nuclear-cytoplasmic localization of interferon regulatory factor 3, a subunit of double-stranded RNA-activated factor 1. Mol. Cell. Biol. 20, 4159–4168. Lai, F., Kazdhan, N., Lichty, B.D., 2008. Using G-deleted vesicular stomatitis virus to probe the innate anti-viral response. J Virol Methods 153, 276–279. Le Bon, A., Etchart, N., Rossmann, C., Ashton, M., Hou, S., Gewert, D., Borrow, P., Tough, D.F., 2003. Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon. Nat. Immunol. 4, 1009–1015. Lin, R., Mamane, Y., Hiscott, J., 1999. Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains. Mol. Cell. Biol. 19, 2465–2474. Lin, R., Noyce, R.S., Collins, S.E., Everett, R.D., Mossman, K.L., 2004. The herpes simplex virus ICP0 RING finger domain inhibits IRF3- and IRF7-mediated activation of interferon-stimulated genes. J. Virol. 78, 1675–1684. Marques, J.T., Devosse, T., Wang, D., Zamanian-Daryoush, M., Serbinowski, P., Hartmann, R., Fujita, T., Behlke, M.A., Williams, B.R., 2006. A structural basis for discriminating between self- and nonself double-stranded RNAs in mammalian cells. Nat. Biotechnol. 24, 559–565. Marques, J.T., Rebouillat, D., Ramana, C.V., Murakami, J., Hill, J.E., Gudkov, A., Silverman, R.H., Stark, G.R., Williams, B.R.G., 2005. Down-regulation of p53 by double-stranded RNA modulates the antiviral response. J. Virol. 79, 11105– 11114. Mosca, J.D., Pitha, P.M., 1986. Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis. Mol. Cell. Biol. 6, 2279–2283. Mossman, K.L., Macgregor, P.F., Rozmus, J.J., Goryachev, A.B., Edwards, A.M., Smiley, J.R., 2001. Herpes simplex virus triggers and then disarms a host antiviral response. J. Virol. 75, 750–758. Mossman, K.L., Smiley, J.R., 2002. Herpes simplex virus ICP0 and ICP34.5 counteract distinct interferon-induced barriers to virus replication. J. Virol. 76, 1995–1998. Mossman, K.L., Upton, C., McFadden, G., 1995. The myxoma virus-soluble interferongamma receptor homolog, M-T7, inhibits interferon-gamma in a species-specific manner. J. Biol. Chem. 270, 3031–3038. Netterwald, J.R., Jones, T.R., Britt, W.J., Yang, S., McCrone, I.P., Zhu, H., 2004. Postattachment events associated with viral entry are necessary for induction of interferon-stimulated genes by human cytomegalovirus. J. Virol. 78, 6688–6691. Ng, P., Graham, F.L., 2002. Construction of first-generation adenoviral vectors. Methods Mol Med 69, 389–414. Nicholl, M.J., Robinson, L.H., Preston, C.M., 2000. Activation of cellular interferonresponsive genes after infection of human cells with herpes simplex virus type 1. J. Gen. Virol. 81, 2215–2218. Noyce, R.S., Collins, S.E., Mossman, K.L., 2006. Identification of a novel pathway essential for the immediate-early, interferon-independent antiviral response to enveloped virions. J. Virol. 80, 226–235. Paladino, P., Cummings, D.T., Noyce, R.S., Mossman, K.L., 2006. The IFN-independent response to virus particle entry provides a first line of antiviral defense that is independent of TLRs and retinoic acid-inducible gene I. J. Immunol. 177, 8008–8016.

399

Peters, K.L., Smith, H.L., Stark, G.R., Sen, G.C., 2002. IRF-3-dependent, NFkappa Band JNK-independent activation of the 561 and IFN-beta genes in response to double-stranded RNA. Proc. Natl. Acad. Sci. U.S.A. 99, 6322–6327. Prescott, J., Ye, C., Sen, G.C., Hjelle, B., 2005. Induction of innate immune response genes by sin nombre hantavirus does not require viral replication. J. Virol. 79, 15007–15015. Preston, C.M., Harman, A.N., Nicholl, M.J., 2001. Activation of interferon response factor-3 in human cells infected with herpes simplex virus type 1 or human cytomegalovirus. J. Virol. 75, 8909–8916. Qin, B.Y., Liu, C., Srinath, H., Lam, S.S., Correia, J.J., Derynck, R., Lin, K., 2005. Crystal structure of IRF-3 in complex with CBP. Structure 13, 1269–1277. Sasai, M., Shingai, M., Funami, K., Yoneyama, M., Fujita, T., Matsumoto, M., Seya, T., 2006. NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic pathways in type I IFN induction. J. Immunol. 177, 8676–8683. Sato, M., Suemori, H., Hata, N., Asagiri, M., Ogasawara, K., Nakao, K., Nakaya, T., Katsuki, M., Noguchi, S., Tanaka, N., Taniguchi, T., 2000. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-[alpha]/[beta] gene induction. Immunity 13, 539–548. Sommereyns, C., Paul, S., Staeheli, P., Michiels, T., 2008. IFN-lambda (IFN-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo. PLoS Pathogen. 4, e1000017. Stojdl, D.F., Lichty, B.D., tenOever, B.R., Paterson, J.M., Power, A.T., Knowles, S., Marius, R., Reynard, J., Poliquin, L., Atkins, H., Brown, E.G., Durbin, R.K., Durbin, J.E., Hiscott, J., Bell, J.C., 2003. VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 4, 263–275. tenOever, B., Servant, M., Grandvaux, N., Lin, R., Hiscott, J., 2002. Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation. J. Virol. 76, 3659–3669. Wathelet, M.G., Berr, P.M., Huez, G.A., 1992. Regulation of gene expression by cytokines and virus in human cells lacking the type-I interferon locus. Eur. J. Biochem. 206, 901–910. Wathelet, M.G., Lin, C.H., Parekh, B.S., Ronco, L.V., Howley, P.M., Maniatis, T., 1998. Virus infection induces the assembly of coordinately activated transcription factors on the IFN-beta enhancer in vivo. Mol Cell 1, 507–518. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira, K., Akira, S., Fujita, T., 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5, 730–737. Zhu, H., Cong, J.P., Shenk, T., 1997. Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: induction of interferon-responsive RNAs. Proc. Natl. Acad. Sci. U.S.A. 94, 13985–13990.

Glossary CBP: CREB-binding protein CMV: cytomegalovirus ERK: extracellular signal-related kinase GAPDH: glyceraldehyde 3-phosphate dehydrogenase GFP: green fluorescent protein HSV: Herpes simplex virus IFN: interferon IKK␧: IkB kinase epsilon IRF-3: interferon regulatory factor 3 ISG: interferon-stimulated gene JAK: Janus kinase JNK: C-Jun N-terminal kinase MAPK: mitogen-activated protein kinase MDA5: melanoma differentiation-associated gene 5 NF␬B: nuclear factor kappa beta PAMP: pathogen-associated molecular pattern pIC: polyinosinic:polycytidylic acid RIG-I: retinoic acid inducible gene I SAPK: stress-activated protein kinase SeV: Sendai virus STAT: signal transducer and activator of transcription TBK-1: Tank binding kinase 1 TLR: Toll-like receptor VSV: vesicular stomatitis virus VV: vaccinia virus