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Journal of Virology, January 2008, p. 609-616, Vol. 82, No. 2
0022-538X/08/$08.00+0     doi:10.1128/JVI.01305-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Establishment and Maintenance of the Innate Antiviral Response to West Nile Virus Involves both RIG-I and MDA5 Signaling through IPS-1 ^,

Brenda L. Fredericksen,^1* Brian C. Keller,^2, Jamie Fornek,^3, Michael G. Katze,³ and Michael Gale Jr.^2,4

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland,¹ Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas,² Department of Microbiology,³ Department of Immunology, University of Washington School of Medicine, Seattle, Washington⁴

Received 14 June 2007/ Accepted 12 October 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
RIG-I and MDA5, two related pathogen recognition receptors (PRRs), are known to be required for sensing various RNA viruses. Here we investigated the roles that RIG-I and MDA5 play in eliciting the antiviral response to West Nile virus (WNV). Functional genomics analysis of WNV-infected fibroblasts from wild-type mice and RIG-I null mice revealed that the normal antiviral response to this virus occurs in two distinct waves. The initial response to WNV resulted in the expression of interferon (IFN) regulatory factor 3 target genes and IFN-stimulated genes, including several subtypes of alpha IFN. Subsequently, a second phase of IFN-dependent antiviral gene expression occurred very late in infection. In cells lacking RIG-I, both the initial and the secondary responses to WNV were delayed, indicating that RIG-I plays a critical role in initiating innate immunity against WNV. However, another PRR(s) was able to trigger a response to WNV in the absence of RIG-I. Disruption of both MDA5 and RIG-I pathways abrogated activation of the antiviral response to WNV, suggesting that MDA5 is involved in the host's defense against WNV infection. In addition, ablation of the function of IPS-1, an essential RIG-I and MDA5 adaptor molecule, completely disabled the innate antiviral response to WNV. Our data indicate that RIG-I and MDA5 are responsible for triggering downstream gene expression in response to WNV infection by signaling through IPS-1. We propose a model in which RIG-I and MDA5 operate cooperatively to establish an antiviral state and mediate an IFN amplification loop that supports immune effector gene expression during WNV infection.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
West Nile virus (WNV) is a member of the Flavivirus genus of the family Flaviviridae, which are enveloped, single-stranded, positive-sense RNA viruses. The recent introduction of WNV into industrialized urban areas in Europe, Israel, and the United States has resulted in epidemics that were associated with a marked increase in both the number of reported cases and the severity of disease among humans and birds (12, 32, 34), suggesting that a more pathogenic strain had emerged. Since its introduction into the United States in 1999, outbreaks of WNV have become yearly occurrences, and the virus has now been detected in nearly every state within the continental United States as well as in parts of Canada, Mexico, and the Caribbean (information found on the CDC website http://www.cdc.gov/ncidod/dvbid/westnile/index.htm) (6). The rapid spread and persistence of WNV indicates that it has firmly established itself in the Western Hemisphere.

The first line of defense against an invading viral pathogen is the innate intracellular antiviral response. The ability to sense an invading pathogen and respond appropriately is a contributing factor in determining the outcome of infection. The cell utilizes a group of proteins known as pathogen recognition receptors (PRRs) to detect the presence of pathogen-associated molecular patterns (PAMPs) within products of viral replication (13, 40). Upon sensing the invading viral pathogen, the cell activates multiple distinct signaling pathways by inducing a number of latent transcription factors (37), which in turn leads to a reprogramming of the cell's gene expression profile and the induction of a wide variety of genes that establish an antiviral state. One such transcription factor, which is central to establishment of the innate antiviral response, is interferon (IFN) regulatory factor 3 (IRF-3) (2). Two classes of PRRs, Toll-like receptor 3 (TLR3) and the helicase family members RIG-I and MDA5, have been shown to stimulate IRF-3 transcriptional activity in response to double-stranded RNA (dsRNA), a well-defined viral PAMP. TLR3, which is expressed on the cell surface or within endocytic vesicles (27, 30), is responsible for detecting extracellular dsRNA, while the cytoplasmic proteins RIG-I and MDA5 sense intracellular dsRNA (44, 45). These interactions initiate signaling cascades that result in the activation of IRF-3 and the subsequent expression of target genes, such as IFN-stimulated gene 54 (ISG54), ISG56, and the beta IFN (IFN-β) (10). It is the expression of these direct IRF-3 target genes that initiate the establishment of an antiviral state to block viral replication. Binding of secreted IFN-β to the type I IFN receptor amplifies the innate antiviral response by triggering the activation of the Janus kinase and signal transducers and activators of transcription (JAK/STAT) signal transduction pathway. The activation of the JAK/STAT pathway leads to the induction of expression of a wide variety of ISGs, which are responsible for conferring the antiproliferative, antiviral, and proapoptotic actions that serve to limit virus infection.

As eukaryotic antiviral programs evolved to combat invading pathogens, viruses have coevolved processes to escape them. The antiviral signaling pathways involved in sensing WNV infection and blocking viral replication have yet to be fully elucidated. We have previously demonstrated that mouse embryo fibroblasts (MEFs) that are deficient in signaling through the TLR3 pathway responded normally to WNV (7). In contrast, ablation of RIG-I delayed the induction of the innate antiviral response compared to what was seen for wild-type (WT) MEFs, indicating that RIG-I mediates the initial detection of WNV infection in cells of nonimmune origin. However, the fact that RIG-I-deficient cells were still capable of responding to WNV suggests that another PRR(s) is also involved in mediating the innate antiviral response. In this report we further assess the role of RIG-I in the establishment of an antiviral state in response to WNV and the involvement of MDA5 in detecting infection.

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Cells and viruses. Low-passage-number primary WT, RIG-I^–/–, and IPS-1^–/– MEFs derived from normal and transgenic C57BL/6 mice, respectively, were kindly provided by Shizuo Akira (16, 19). WT and MDA5^–/– primary MEFs derived from C57BL/6 mice were obtained from Marco Colonna (9). All cell lines were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, antibiotic-antimycotic solution, and nonessential amino acids (complete DMEM). U-2 OS/NS3/4A cells were propagated in complete DMEM supplemented with 500 mg/ml G418, 1 mg/ml puromycin, and 1 mg/ml tetracycline (42). Working stocks of WNV-TX 2002-HC (TX02) and WNV-NY, which are recent lineage I isolates with identical growth characteristics and antiviral responses in vitro (17), were generated by passaging the virus one time on 293 cells at a low multiplicity of infection (MOI), and aliquots were stored at –80°C. Titers of working viral stocks on U-2 OS/NS34A cells were determined by plaque assay. Viral titers on the various MEF cell lines were determined using a focus-forming assay as previously described with Vector VIP (Vector Laboratories) as a substrate for horseradish peroxidase (8). Titers for WNV were similar for WT and RIG-I^–/– MEFs, which allowed cultures to be comparably infected. The amount of virus added to cultures to achieve the indicated MOI was calculated using the titer of the viral stock on the respective cell line unless otherwise stated. Sendai virus (SenV) Cantrell strain (Charles River) was amplified in chicken embryos.

Expression microarray format and data analysis. Cultures of WT and RIG-I^–/– MEFs in six-well plates were infected with TX02 at an MOI of 0.5 in duplicate. At the indicated time points, total RNA was extracted from cells by use of TRIzol reagent as recommended by the manufacturer (Invitrogen Life Technologies, Inc.). Total RNA samples were amplified using a RiboAmp RNA amplification kit (Arcturus) as described by the manufacturer. The quality of amplified RNA was evaluated by capillary electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies). Microarray format, protocols for probe labeling, and array hybridization are described at http://expression.viromics.washington.edu. Briefly, a single experiment comparing two mRNA samples was done with four replicate mouse (V2) 22K oligonucleotide expression arrays (Agilent Technologies) using the dye label reverse technique. This allows for the calculation of mean ratios between expression levels of each gene in the analyzed sample pair, standard deviation, and P values for each experiment. Spot quantitation, normalization, and application of a platform-specific error model was performed using Agilent's Feature Extractor software, and all data were then entered into a custom-designed database, Expression Array Manager, and then uploaded into Rosetta Resolver System 6.0 (Rosetta Biosoftware) and Spotfire DecisionSite for Functional Genomics 8.1 (Spotfire). Data normalization and the Resolver error model are described on the website http://expression.viromics.washington.edu. This website is also used to publish all primary data in accordance with the proposed MIAME standards (4). Selection of genes for data analysis was based on a change (n-fold) of twofold or greater.

Antibodies. Rabbit anti-human ISG56, rabbit anti-mouse ISG54, rabbit anti-mouse ISG56, and rabbit anti-human ISG15 antibodies were kindly provided by Ganes Sen. Goat anti-SenV, mouse anti-hepatitis C virus (HCV) NS3/4A, and rabbit anti-mouse IRF-3 were purchased from Biodesign, Novocastra Laboratories, and Zymed, respectively. Mouse anti-WNV was obtained from the CDC, and rabbit anti-human IRF-3 serum was kindly provided by Michael David. Goat anti-GAPDH and mouse anti-GAPDH were purchased from Santa Cruz and Abcam, respectively. Peroxidase-conjugated goat anti-rabbit and donkey anti-goat were purchased from Jackson ImmunoResearch. Goat anti-mouse conjugated to peroxidase was obtained from Novocastra. For immunofluorescence assays, donkey anti-goat immunoglobulin G (IgG)-Alexa 594, goat anti-rabbit IgG-Alexa 488, and goat anti-mouse IgG-Alexa 350 were purchased from Molecular Probes, and goat anti-mouse IgG-rhodamine antibody conjugate was purchased from Jackson ImmunoResearch.

Immunoblot analysis. Cells were lysed in radioimmunoprecipitation assay buffer (10 mM Tris, 150 mM NaCl, 0.02% Na-deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate) containing protease inhibitors (Sigma) and okadaic acid (1 mM) (Sigma). Proteins (20 µg) were resolved on 10% polyacrylamide gels containing sodium dodecyl sulfate. After electrophoresis, proteins were transferred to NitroPure nitrocellulose transfer membranes (Micron Separations Inc.), and blots were blocked overnight at 4°C. Blots were probed with the indicated primary antibodies and appropriate conjugated secondary antibodies. Protein bands were visualized using the ECL+ Western blotting detection reagents (Amersham Biosciences) followed by exposure of the blot to film.

Indirect immunofluorescence analysis (IFA). The indicated cell lines were grown on tissue culture chamber slides and infected with either WNV-NY or SenV (50 hemagglutinating units). At the indicated times postinfection, slides were washed with phosphate-buffered saline (PBS) and fixed with 3% paraformaldehyde for 30 min at room temperature. Cell monolayers were permeabilized with a solution of PBS-0.2% Triton X-100 for 15 min, followed by a 1-h incubation in PBS containing 10% normal goat serum. After being rinsed with PBS, MEFs were incubated for 1 h in the presence of rabbit anti-mouse IRF-3 (1:500), mouse polyclonal anti-WNV antibody (1:750), and goat polyclonal anti-SenV (1:500) in PBS-0.05% Tween 20-3% bovine serum albumin. Cells were washed three times with PBS-0.5% Tween 20 and incubated with donkey anti-goat IgG-Alexa 594 antibody conjugate (1:2,000) for 1 h at room temperature. Cells were washed three times and incubated with goat anti-rabbit IgG-Alexa 488 (1:2,000) and goat anti-mouse IgG-Alexa 350 (1:1,000) for 1 h. Permeabilized U-2 OS/NS3/4A cells were incubated with a rabbit polyclonal anti-human IRF-3 (1:500) and a mouse anti-HCV NS3/4A (1:250) in PBS-0.05% Tween 20-3% bovine serum albumin and washed three times with PBS-0.5% Tween 20. Slides were then incubated with goat anti-rabbit IgG-Alexa 488 antibody conjugate and goat anti-mouse IgG-rhodamine (1:2,000) for 1 h at room temperature. Following incubation with secondary antibodies, cells were washed three times with PBS-0.5% Tween 20 and allowed to dry, and the slides were overlaid with Vectashield solution (Vector Labs). The coverslips were mounted and visualized with a Zeiss Axiovert fluorescence microscope equipped with a digital camera.

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RIG-I-mediated regulation of the innate antiviral response to WNV. We have previously demonstrated that ablation of RIG-I delays the induction of a subset of ISGs in response to WNV infection, suggesting that RIG-I signaling is required to trigger the initial antiviral response to WNV (7). In order to examine the global effect of RIG-I ablation on the cell's ability to respond to WNV, gene expression profiles of WNV-infected WT and RIG-I^–/– MEFs were assessed using microarray analysis. Cells were infected at an MOI of 0.5 to allow cell-to-cell spread of the virus within the cultures and thereby more closely approximate an in vivo infection. Total RNA was collected at 20 h postinfection, an early time point at which the antiviral response has been shown to be detectable in WT but not RIG-I-deficient MEFs, and 32 h postinfection, a time point at which a subset of ISGs have previously been shown to be induced in WNV-infected WT and RIG-I^–/– MEFs (7). These two time points were chosen in an attempt to distinguish between antiviral genes with delayed induction profiles in WNV-infected RIG-I^–/– MEFs and genes whose expression is completely dependent on RIG-I signaling. Comparison of the gene expression profiles for WT and RIG-I^–/– MEFs demonstrated that the global response to WNV infection was attenuated at 20 h postinfection in RIG-I-deficient MEFs (Fig. 1). Only 0.3% of genes were differentially regulated in WNV-infected RIG-I^–/– MEFs compared to 3.3% of genes in WT MEFs. In contrast, similar percentages of genes were differentially regulated in RIG-I^–/– and WT MEFs at 32 h postinfection, demonstrating that signaling through RIG-I is essential for stimulation of the initial antiviral response to WNV as well as a broad modulation of the cell's transcriptional profile.

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FIG. 1. Microarray analysis of WT and RIG-I–/– MEFs. Graphic representation of the total number of host cell genes whose expression was differentially modulated in WNV-infected cells compared to mock-infected cultures. Genes were selected based on two criteria: a greater-than-99% probability of being differentially expressed (P < 0.01) and an overall change in expression of twofold or greater. The total number and frequency of genes that fit these criteria are indicated below each bar.

A focused analysis of known immune response genes identified 77 WNV-responsive genes in WT MEFs, all of which exhibited sustained expression throughout the course of the experiment (Fig. 2). Of these genes, 24 exhibited similar kinetics of induction in both WT and RIG-I^–/– MEFs (Fig. 2A; also see Table S1 in the supplemental material), though several genes exhibited a reduced level of induction in RIG-I^–/– MEFs. The induction patterns of the remaining WNV-responsive genes were substantially altered in the absence of RIG-I (Fig. 2B and C; also see Tables S2 and S3 in the supplemental material). Two additional expression profiles were observed for the WNV-responsive genes in RIG-I^–/– cells. The first profile consisted of genes with delayed kinetics of induction (Fig. 2B; also see Table S2 in the supplemental material), similar to the pattern previously reported for ISG54 and ISG56 (7). Expression of these genes was detected only at the 32-h-postinfection time point in RIG-I^–/– MEFs; however, several of these genes, such as the flavivirus resistance gene OAS1b, were expressed at lower levels in RIG-I^–/– MEFs than in WT MEFs. Microarray analysis also identified a novel subset of WNV-responsive genes that were not induced in RIG-I-deficient MEFs at either time point (Fig. 2C; also see Table S3 in the supplemental material).

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FIG. 2. Expression profiles of WNV-responsive genes. (A) WNV-responsive genes with similar kinetics of induction in WT and RIG I–/– MEFs. (B) Genes exhibiting a delayed induction of expression in RIG-I–/– MEFs compared to WT MEFs. (C) Genes that were not induced in RIG-I–/– MEFs compared to mock-infected cells at either time point.

IFN- mediates a second phase of ISG expression in response to WNV infection. Among the WNV-responsive genes detected in WT MEFs were several subtypes of IFN- (Fig. 2B), the expression of which would be expected to initiate an amplification of the innate antiviral response. In the case of WNV-infected RIG-I^–/– cells, the expression of IFN- was delayed until approximately 32 h postinfection (Fig. 2B). This delay in IFN- expression would be predicted to postpone the activation of the IFN amplification loop and the subsequent second round of induction of ISGs. This suggested that the WNV-responsive genes that were not expressed in RIG-I^–/– MEFs by 32 h postinfection (Fig. 2C) might be induced by IFN- at later times. In order to assess this possibility, the kinetics of induction of ISG15, an IFN-inducible gene that has been previously shown to be induced in response to WNV infection in a variety of cells in vitro and tissues in vivo (8, 18, 41), was examined by Western blot analysis (Fig. 3A). As a control, the expression of ISG54, a protein with an expression profile similar to that of IFN- in both WT and RIG-I^–/– MEFs, was also monitored. As previously reported, WNV-mediated induction of ISG54 expression was delayed in RIG-I^–/– MEFs compared to WT; however, expression was clearly detectable in both cell lines by 32 h postinfection (7). In comparison to ISG54, ISG15 exhibited an extended lag period prior to the induction of expression in RIG-I-deficient cells.

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FIG. 3. Analysis of the induction kinetics of WNV-responsive genes at late times postinfection. (A) Comparison of the kinetics of expression of ISG54 and ISG15 in WT and RIG-I–/– MEFs infected with WNV. WT and RIG-I–/– MEFs were infected with WNV and whole-cell lysates were collected at the indicated times postinfection (p.i.). Steady-state levels of ISG54, ISG15, WNV, and GAPDH were examined by Western blotting. (B) WT and RIG-I–/– MEFs were infected with WNV in the presence of control -IgG or -IFN-/β antibodies (400 U/ml). Whole-cell lysates were assessed for steady-state levels of ISG15, STAT1, and GAPDH. -, anti.

The timing ISG15 expression suggested that its induction was mediated through the IFN- amplification loop. To assess the role of IFN- in WNV-induced ISG expression, WT and RIG-I^–/– MEFs were infected with WNV in the presence of anti-IFN-/β or control antibodies, and the expression of ISG15 was examined by Western blotting at late times postinfection (Fig. 3B). Anti-IFN-/β antibodies reduced the level of WNV-induced ISG15 expression in WT MEFs and blocked expression in RIG-I^–/– MEFs, while control antibodies had no effect on the induction of ISG15. To confirm that this observation was not restricted to ISG15, the expression pattern of a second WNV-responsive gene, STAT1, was also examined. STAT1 was not detected in WNV-infected RIG-I^–/– MEFs by microarray analysis at 32 h postinfection (Fig. 2C). However, Western blot analysis confirmed that STAT1 was induced in RIG-I-deficient cells at later times postinfection (Fig. 3B), confirming that a second round of innate antiviral gene expression is induced during WNV infection. Furthermore, the addition of anti-IFN-/β antibodies to culture supernatants inhibited the induction of STAT1, demonstrating that induction of a second wave of WNV-responsive gene expression is mediated through IFN.

MDA5 mediates IRF-3 activation in the absence of RIG-I. The fact that cells lacking RIG-I were still able to respond to WNV infection indicated that a second PRR was also involved in detecting viral replication. A likely candidate for a secondary PRR molecule sensor for WNV infection is the RIG-I homolog MDA5. In initial experiments with MEFs derived from MDA5^–/– and littermate control mice, the rate of WNV replication was substantially reduced compared to that previously observed for RIG-I^–/– and control WT MEFs (compare Fig. 3A and 4). The low level of WNV replication in WT and MDA5-deficient cells failed to stimulate a robust antiviral response (Fig. 4). Therefore, MDA5's potential contribution to the stimulation of the antiviral response to WNV infection could not be accurately assessed using these cell lines. In order to overcome this issue, WNV-mediated induction of the innate antiviral response was evaluated for RIG-I^–/– MEFs coinfected with SenV. It has previously been demonstrated that the V proteins of several paramyxoviruses, including SenV, specifically bind to MDA5 and subsequently block its signaling activity (1, 5). Therefore, signaling through the MDA5 pathway can be selectively inhibited by infecting cells with SenV. RIG-I^–/– MEFs were infected with SenV or WNV or coinfected with SenV and WNV, and the innate host response was assessed by monitoring the cellular localization of IRF-3 by IFA (Fig. 5). As previously reported, infection with WNV alone induced IRF-3 nuclear localization (Fig. 5g) (7). In contrast, SenV infection did not induce IRF-3 activation in RIG-I^–/– MEFs (Fig. 5f), confirming that the virus was obstructing signaling through the MDA5 pathway. Similarly, IRF-3 nuclear localization was not detected in cultures coinfected with SenV and WNV (Fig. 5h), demonstrating that SenV blocked the cell's ability to respond to WNV infection. These data indicated that the innate antiviral response to WNV in RIG-I-deficient cells is mediated through MDA5.

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FIG. 4. WNV infection of MDA5–/– and WT MEFs. Whole-cell lysates collected at the indicated times postinfection were analyzed for steady-state levels of ISG54, WNV protein, and GAPDH by Western blot analysis.

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FIG. 5. Cellular localization of IRF-3 in RIG-I–/– MEF coinfected with WNV and SenV. Mock (a, e, and i), SenV (b, f, and j), and WNV (c, g, and k) cultures, along with cultures coinfected with SenV and WNV (d, h, and l), were fixed at 72 h postinfection and probed for WNV protein expression (a to d), IRF-3 (e to h), and SenV protein expression (i to l). -, anti.

IPS-1 mediates the innate antiviral response to WNV. Signaling through the RIG-I and MDA5 pathways converges at the adaptor molecule IPS-1 (16, 28, 38, 43). If the RIG-I and MDA5 pathways were solely responsible for sensing WNV infection, then IPS-1 would be predicted to play a central role in establishing an antiviral state in response to infection. It has previously been demonstrated that the NS3/4A protease of HCV prevents signaling through both the RIG-I and MDA5 pathways by cleaving IPS-1 (20, 23, 28). Therefore, HCV NS3/4A was used as a tool to investigate the involvement of IPS-1 in WNV-mediated activation of the innate antiviral response. The cellular distribution of IRF-3 was monitored in cultures infected with WNV in the presence or absence of HCV NS3/4A. In control cells, which were not expressing HCV NS3/4A, WNV induced nuclear translocation of IRF-3 as previously reported (Fig. 6A, panel a) (8). Conversely, in the presence of HCV NS3/4A, WNV-mediated translocation of IRF-3 was completely inhibited, suggesting that HCV NS3/4A blocked the cells' ability to respond to WNV infection. Furthermore, HCV NS3/4A expression also prevented induction of the IRF-3 target gene ISG56 in response to WNV infection (Fig. 6B).

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FIG. 6. IPS-1 plays a central role in modulating the innate antiviral response to WNV. (A) Induction of IRF-3 nuclear localization by WNV infection. Cellular localizations of IRF-3 (panels a and b) and HCV NS3/4A (panels c and d) protein expression were examined by IFA in U-2 OS/NS3/4A cells propagated in the presence (panels a and c) or absence (panels b and d) of tetracycline. (B) Effect of HCV NS3/4A on WNV-induced expression of IRF-3 target genes. U-2 OS/NS3/4A cells propagated in the presence or absence of tetracycline were infected with WNV (MOI = 3). Induction of ISG56 was assessed by Western analysis of whole-cell lysates harvested at the indicated times postinfection. Blots were stripped and reprobed for HCV NS3/4A to confirm the induction of expression in the absence of tetracycline. The levels of GAPDH expression were also assessed to control for loading. (C) Activation of the innate antiviral response in IPS-1–/– MEFs. WT and IPS-1–/– MEFs were infected at an MOI of 5 (based on titer from Vero cells), and whole-cell lysates were collected at 48 h postinfection. Expression of ISG54, ISG56, WNV, and GAPDH was assessed by Western blotting.

The role of IPS-1 in signaling the induction of the innate antiviral response to WNV was also assessed using MEFs recovered from IPS-1-deficient mice. Lysates recovered from WNV-infected WT and IPS-1^–/– MEFs were examined for the expression of ISG56 and a second IRF-3 responsive gene, ISG54 (Fig. 6C). In the absence of IPS-1, WNV-mediated induction of both ISG56 and ISG54 was completely ablated, demonstrating that signaling through IPS-1 is necessary for the establishment of an antiviral state in response to WNV infection.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The rapid induction of the innate antiviral response is critical for controlling viral replication, thus providing time for the adaptive arm of the immune system to establish an effective response. We have previously demonstrated that ablation of RIG-I enhanced peak viral titers of WNV despite the fact that the cells were still capable of mounting an innate antiviral response (7). One possible explanation for the RIG-I-dependent enhancement of resistance to WNV-NY infection is that signaling through RIG-I induces a specific subset of antiviral genes that are more effective at inhibiting viral replication. Alternatively, the kinetics of induction of antiviral genes may be critical for controlling WNV. To distinguish between these two possibilities, microarray analysis was used to compare the gene expression profiles of WNV-infected WT and RIG-I^–/– MEFs. A focused analysis of immunomodulatory genes identified 77 genes that were differentially regulated in WT MEFs in response to WNV infection. Of these genes, 24 exhibited similar kinetics of induction in WT and RIG-I^–/– MEFs, though is should be noted that the majority of these genes were expressed at reduced levels in RIG-I MEFs at at least one time point.

The remaining WNV-responsive genes were divided into two additional subsets of genes. The first subset of genes exhibited a delayed induction of expression in the absence of RIG-I, which was similar to the expression profiles previously determined for ISG54 and ISG56 by use of quantitative real-time reverse transcription-PCR and Western blot analysis (7). This expanded analysis identified 25 additional genes with delayed induction, several of which also exhibited reduced levels of expression at 32 h postinfection (Fig. 2B; also see Table S2 in the supplemental material). Among the genes whose expression was delayed in the absence of RIG-I was the transcription factor IRF-7, which was previously shown to be induced in vivo in response to WNV infection (41). IRF-7 functions as a direct transducer of virus-induced signaling and plays a critical role in amplifying the antiviral response. Furthermore, IRF-7-mediated induction of the innate antiviral response has been shown to result in an extensive remodeling of the cell's transcriptional profile (3). This suggests that the delayed global response to WNV infection in RIG-I^–/– MEFs may be due in part to the delayed expression of IRF-7.

As in previous studies, several members of the inducible oligoadenylate synthetase (OAS) family were determined to be upregulated in response to WNV infection by microarray analysis studies (18, 35, 41). OASs are dsRNA-dependent enzymes that catalyze the polymerization of ATP into 2'-5'-linked oligoadenylates, which in turn bind and activate the latent endoribonuclease RNase L. Once activated, RNase L blocks viral replication and enhances the antiviral response by degrading viral and cellular RNAs (25). Recent studies have shown that the 2'-5'OAS/RNase L pathway plays an important role in controlling WNV replication in both resistant and susceptible mouse cell lines (14, 24, 35). Therefore, it is likely that the enhanced replication of WNV in RIG-I-deficient MEFs is at least partially due to the delayed and/or reduced expression of several members of the OAS/RNase L system. However, it should be pointed out that the B57BL/6 mouse strains from which both WT and RIG-I^–/– MEFs were derived encode a point mutation within the flavivirus resistance gene, Oas1b, that results in a premature stop codon (26, 31). Therefore, since both WT and RIG-I^–/– MEFs produce a truncated inactive from of Oas1b it is unlikely that the delayed expression of Oas1b in RIG-I^–/– MEFs contributed to the observed increase in viral replication.

Functional genomic analysis also identified an additional subset of genes that were not induced by WNV infection in RIG-I-deficient MEFs at either time point. However, this subset consisted of IFN-inducible genes whose expression would be predicted to be induced by IFN-. WNV has previously been shown to block signaling through the JAK/STAT pathway in response to exogenous IFN- (11, 17, 22, 36). Therefore, one possible explanation for the lack of expression of these IFN-inducible genes in RIG-I-deficient MEFs was that the delayed expression of IFN- in these cells allowed WNV sufficient time to impose a complete blockage on JAK/STAT signaling, thereby preventing IFN-mediated induction of secondary ISGs. Alternatively, the delayed expression of IFN- in RIG-I^–/– MEF could simply result in a subsequent prolonged delay in the induction of a second wave of ISG expression. To distinguish between these two possibilities, we examined the induction of ISG15 and STAT1, two ISGs that previously have been shown to be upregulated in response to WNV infection (8, 18, 35, 41) at time points after IFN- expression was detected. Neither protein was detected in RIG-I^–/– MEFs until approximately 40 h postinfection, demonstrating that there was a prolonged delay prior to induction of these ISGs. Furthermore, anti-IFN-/β antibodies ablated the expression of both proteins in RIG-I^–/– MEFs, demonstrating that IFN mediates an induction of a second wave of WNV-responsive genes. These data suggest that WNV does not impose a complete blockade on IFN-dependent pathways but rather attenuates signaling in response to IFN. Combined, our data suggest that increased viral replication in RIG-I^–/– MEFs is due to the delayed and/or reduced expression of antiviral genes rather then to a complete ablation of expression of critical WNV-responsive genes.

As with the adaptive immune response, the innate antiviral response must be able to distinguish self from nonself. This discrimination is mediated in part by specialized PRRs that recognize specific PAMPs produced during the course of infection, thereby triggering signaling events that initiate an innate immune response against the pathogen. In the case of RNA viruses, cells have evolved to recognize dsRNA structures that are the by-product of viral replication. The significance of dsRNA in cellular detection of viral infection is attested to by the fact that multiple PRRs, including RIG-I, MDA5, and TLR3, recognize this specific PAMP.

Previous studies have indicated that RIG-I and MDA5 are not functionally redundant sensors; rather, each PPR recognizes a specific subset of viruses (15). Initial studies indicated that RIG-I is responsible for detecting negative-strand RNA viruses, such as paramyxoviruses, influenza virus, and vesicular stomatitis virus, as well as two related positive-strand viruses, Japanese encephalitis virus and HCV (15, 33, 39). In contrast, MDA5 has been show to mediate recognition of another positive-strand RNA virus, encephalomyocarditis virus, a prototypic member of the picornavirus family (9, 15). We have previously demonstrated that RIG-I plays an essential role in the initial detection of WNV (7). However, functional genomic analysis confirmed that the deletion of RIG-I does not result in the ablation of the antiviral response to WNV but rather in a delay in the expression of WNV-responsive antiviral genes. This suggests that another PRR(s), in addition to RIG-I, is involved in signaling activation of the innate antiviral response. Our data indicate that MDA5 serves as a secondary PRR during WNV infection, functioning in the amplification of the innate immune response initiated by RIG-I. PRR signaling in response to WNV infection was completely dependent upon IPS-1, confirming that RIG-I and MDA5 signal downstream gene expression through the IPS-1 adaptor protein. Therefore, in contrast to what has been previously reported for other viruses, in which RIG-I or MDA5 independently triggers innate immunity (9, 15, 39), RIG-I and MDA5 function cooperatively to establish an antiviral state in response to WNV infection.

Based on these data, we propose that the antiviral response to WNV is comprised of two distinct waves of ISG induction (Fig. 7). As for many other viruses, RIG-I is the initial sensor responsible for detecting WNV infections. Once activated, RIG-I triggers a signaling cascade that results in the activation of latent transcription factors, such as IRF-3. These transcription factors in turn induce the expression of antiviral genes and the establishment of an initial antiviral state within the infected cell. MDA5 and several IFN- subtypes, which are essential for the induction and maintenance of a secondary antiviral response, are among the WNV-responsive genes upregulated during the initial wave of ISG expression (Fig. 2). During the secondary antiviral phase, MDA5 and RIG-I work in concert to maintain the induction of the antiviral genes, while IFN- functions to amplify and/or expand the response in an attempt to control viral replication. WNV, in turn, has evolved multiple mechanisms to circumvent the antiviral effects of these pathways. We have previously demonstrated that virulent strains of WNV evade detection by the host cells at early times postinfection, which allows these strains to replicate to high levels early in infection (7). However, once a productive infection has been established, the antiviral pathways become activated and WNV must then utilize a second mechanism to control the cellular environment. Several WNV proteins have previously been shown to impede signaling through the JAK/STAT pathway (21, 29). Therefore, expression of high levels of these proteins late in infection would presumably attenuate JAK/STAT signaling and thereby prevent the induction of a complete antiviral response. By combining multiple mechanisms, WNV successfully controls both the kinetics of induction and the overall gene expression profile of the innate antiviral response, thus enabling the virus to establish a productive infection.

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FIG. 7. Model of the innate antiviral response to WNV. Details are described in the text.

 
We thank S. Akira, T. Fujita, G. Sen, M. David, N. Kato, M. Colonna, and P. Y. Shi for reagents.

This work was supported by NIH grant AI57568 (M.G.) and NIH grant K22AI063028 (B.L.F.).

 
* Corresponding author. Mailing address: 2101 Microbiology, College Park, MD 20742. Phone: (301) 405-1251. Fax: (301) 314-9489. E-mail: bfreder{at}umd.edu

Published ahead of print on 31 October 2007.

Supplemental material for this article may be found at http://jvi.asm.org/.

B. C. Keller and J. Fornek contributed equally to this study.

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Journal of Virology, January 2008, p. 609-616, Vol. 82, No. 2
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