IFN Regulatory Factor-1 Bypasses IFN-Mediated Antiviral Effects through Viperin Induction

This information is current as Anja Stirnweiss, Antje Ksienzyk, Katjana Klages, Ulfert of October 1, 2021. Rand, Martina Grashoff, Hansjörg Hauser and Andrea Kröger J Immunol 2010; 184:5179-5185; Prepublished online 22 March 2010; doi: 10.4049/jimmunol.0902264 http://www.jimmunol.org/content/184/9/5179 Downloaded from

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Material 4.DC1 http://www.jimmunol.org/ References This article cites 35 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/184/9/5179.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IFN Regulatory Factor-1 Bypasses IFN-Mediated Antiviral Effects through Viperin Gene Induction

Anja Stirnweiss,1 Antje Ksienzyk, Katjana Klages,2 Ulfert Rand, Martina Grashoff, Hansjo¨rg Hauser, and Andrea Kro¨ger

Viperin is an antiviral whose expression is highly upregulated during viral infections via IFN-dependent and/or IFN- independent pathways. We examined the molecular alterations induced by the transcriptional activator IFN regulatory factor (IRF)-1 and found viperin to be among the group of IRF-1 regulated . From these data, it was not possible to distinguish genes that are primary targets of IRF-1 and those that are targets of IRF-1–induced , like IFN-b. In this study, we show that IRF-1 directly binds to the murine viperin promoter to the two proximal IRF elements and thereby induces viperin expression. Infection studies with embryonal fibroblasts from different gene knock-out mice demonstrate that IRF-1 is essential, whereas the type I IFN system is dispensable for vesicular stomatitis virus induced viperin gene transcription. Further, IRF-1, but Downloaded from not IFN type I, mediates the induction of viperin transcription after IFN-g treatment. In contrast, IRF-1 is not required for IFN- independent viperin induction by Newcastle disease virus infection and by infection with a vesicular stomatitis virus mutant that is unable to block IFN expression and secretion. We conclude that the IRF-1 mediated type I IFN independent mechanism of enhanced viperin expression provides a redundant mechanism to protect cells from viral infections. This mechanism becomes important when viruses evade innate immunity by antagonizing the induction and function of the IFN system. The Journal of Immunology, 2010, 184: 5179–5185. http://www.jimmunol.org/

he early innate immune system defends against infections and myxovirus resistance gene 1 are nevertheless able to mount by eliciting a nonspecific response after the recognition of a substantial, residual antiviral response (2). For the majority of T various pathogen-associated molecular patterns. TLRs and ISG-encoded proteins, the exact molecular mechanisms un- members of the retinoic acid inducible gene family recognize derlying their antiviral actions remain unknown. foreign nucleic acids within endosomal and cytoplasmic com- Viperin (virus inhibitory protein, endoplasmatic reticulum- partments, initiating a signaling cascade that involves the induction associated, IFN-inducible), an highly conserved antiviral ISG, was of type I IFN through the transcription factors IFN regulatory factor initially identified as a human CMV (HCMV)-inducible protein in by guest on October 1, 2021 (IRF) 3 and NFkB (1). IFNs represent the first line of defense primary skin cultures (3). Its expression is highly upregulated by against viral infection and mediate their antiviral activities by the type I, II and III IFNs (4–6), microbial products like LPS (7–9), induction of IFN-stimulated genes (ISGs). These IFN-stimulated and by infection with a broad range of viruses (4, 8–12), sug- mediators interfere with many steps of the viral replication process gesting an important role in the innate immune response. Fur- through mechanisms that are extremely complex and involve thermore, data from viperin-deficient mice indicate that viperin is multiple overlapping or related pathways. For instance, mice that required for optimal Th2 response and thus is involved in adaptive are triply deficient for RNase L, RNA-dependent protein kinase R, immune responses as well (13). Viperin interferes with the prop- agation of a broad spectrum of viruses, for example, HCMV, HIV, hepatitis-C virus (HCV), alfavirus, and influenza A virus (10, 11, Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection 14–16). In HCMV infected cells, the expression of viperin inhibits Research, Braunschweig, Germany the expression of several structural proteins that are critical for 1Current address: Institute of Molecular and Clinical Immunology, Otto-von-Guericke viral assembly and maturation (10). In influenza A virus-infected University of Magdeburg, Magdeburg, Germany. cells, viperin prevents virus release from the plasma membranes 2 Current address: Department of Experimental Immunology, Helmholtz Centre for by influencing the farnesyl diphospate synthetase, which is re- Infection Research, Braunschweig, Germany. sponsible for the organization of lipid rafts (11). Recently, viperin Received for publication July 15, 2009. Accepted for publication February 22, 2010. had been shown to reduce the secretion of soluble, but not This work was supported by the German Research Foundation (SFB 566, B7), the membrane-bound proteins and to alter the endoplasmatic re- German Ministry of Research and Education (FORSYS-Partner), Deutsche Kreb- shilfe (10-2136), and Wilhelm Sander Stiftung. ticulum membrane structure (17). How this contributes to the Address correspondence and reprint requests to Dr. Andrea Kro¨ger, Department of antiviral capability of viperin remains unclear. Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, In- Given the critical role of innate immune response in survival from hoffenstraße 7, D-38124 Braunschweig, Gemany. E-mail address: andrea.kroeger@ infection, it is not surprising that a number of molecules involved in helmholtz-hzi.de virus recognition and IFN signaling, for example,IRF-3 and IFN itself The online version of this article contains supplemental material. are affected by viral proteins (18). Cells infected with Japanese en- Abbreviations used in this paper: CHX, cycloheximide; HCMV, human cytomegalo- virus; HCV, hepatitis-C virus; IRF, IFN regulatory factor; IRF-E, IRF element; ISG, cephalitis virus fail to clear the virus infection, because through a yet IFN stimulated gene; ISGF3, ISG factor 3; ISRE, IFN stimuated regulatory element; unknown mechanism thevirus evades the antiviral effect of viperin by MEF, mouse embryonic fibroblasts; MOI, multiplicity of infection; NDV, Newcastle degrading the protein (12). The existence of these viral mechanisms disease virus; VSV, vesicular stomatitis virus; wt, wild-type. forced evolution to develop redundant pathways for detection and Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 inhibition of viral infections. The findings of several groups revealed www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902264 5180 IFN INDEPENDENT, IRF-1 DEPENDENT VIPERIN INDUCTION that the activation of viperin expression can result through IFN- Luciferase Kit (Promega, Madison, WI). Signals were normalized to the independent mechanisms, depending on the infecting virus (8, 9, 12). Renilla luciferase activity. All experiments were performed at least twice In this study, we describe the transcription factor IRF-1 as an essential with triplicates before calculating mean values and standard deviations. mediator of viperin induction, acting through an IFN-independent Electro mobility shift assay pathway in vesicular stomatitis virus (VSV) infected cells. We also Murine IRF-1 and firefly luciferase were in vitro translated using the TNT investigated the molecular basis for the IRF-1 induced promoter ac- T7 Quick Coupled Transcription/Translation System (Promega), according tivation. Furthermore, we show that an enhanced viperin expression, to the manufacturer’s protocol. pMT7-IRF-1 and luciferase T7 control DNA comparable to that in VSV infected cells, results in a reduction of served as templates. EMSA experiments were performed with 5 mlinvitro VSV replication even in the absence of functional IFN signaling. translated protein mix in the absence or presence of 1 mganti-IRF-1(M-20; Santa Cruz Biotechnology, Santa Cruz, CA), as described in Klar and Bode (22). The used oligonucleotides were shown in the Supplemental Table II. Materials and Methods Cell culture and treatments IFN assay IFNAR2/2, IRF-12/2, and STAT12/2 mouse embryonic fibroblasts (MEFs) Type I IFN concentration in the cell culture supernatants was determined by an antiviral assay using LMTK2 cells as described previously (23). Briefly, were kindly provided by R. Zawatzky (Deutshes Krebsforschungszentrum, 2 Heidelberg, Germany), T. Decker (University of Vienna, Vienna, Austria), we seeded LMTK cells in 96 wells and treated them for 24 h with serial and M. Mu¨ller (Veterinary University of Vienna, Vienna, Austria). MEFs, diluted supernatants. Cells were challenged with VSV,IFN in the supernatant NIH3T3, and myc/rasNIH3T3IH cells (19) were grown in DMEM supple- protects cells from the cytopathic effect through viral infection. Effects from mented with 10% FCS and antibiotics. IRF-1hER fusion protein was acti- supernatants were compared with standard type I IFN effect (PBL). vated by 1 mM b-estradiol (Serva, Heidelberg, Germany). Cells were stimulated with 500 U/ml IFN-b or 100 U/ml IFN-g (PeproTech, Rocky Hill, Promoter analysis Downloaded from NJ) for the indicated time periods. Treatment with cycloheximide (CHX) To predict potential transcription factor binding sites in the murine viperin (60 mg/ml) occurred 2 h before IRF-1hER activation and IFN-g stimulation. promoter, the sequence from 21814 to +100 was analyzed by the web tool Viral infection TESS (www.cbil.upenn.edu/tess). This program uses sites or consensus strings and positional weight matrices from the TRANSFAC, IMD, and the Cells were infected with Newcastle disease virus (NDV), VSV (Indiana strain), CBIL-GibbsMat database. or AV2 for 1 h in DMEM without FCS and harvested at the indicated time points postinfection. AV2 is a VSV strain containing a mutated M-protein. For Western blot analysis and Abs http://www.jimmunol.org/ virus challenge, NIH3T3 cells were transiently transfected with pVBC-3vigGN Western blot analysis was accomplished according to standard procedures or a control plasmid using Metafectene (Biontex, Martinsried, Germany). Cells using ECL detection (Amersham Munich, Germany). The following pri- were infected 24 h after transfection, and the supernatant was collected 24 h 2 mary Abs were used: IRF-3P (Ser396) (4D4G), Cell Signaling (Danvers, postinfection. LMTK cells were infected with serial dilutions of the super- MA); VSV-G (P5D4), Boehringer Mannheim (Mannheim, Germany); and natants to determine viral titers (PFU/ml) by a plaque-forming assay. actin (Ab-1), Oncogene (Cambridge, United Kingdom). HRP–conjugated Vector constructs anti-rabbit and anti-mouse Abs (Amersham) were used as secondary Abs. The 21814/+14 viperin-promoter reporter construct was created by am- plifying 1800 bp of the murine viperin promoter from IB10 cells (Primer: Results 59-AAT TCC GCT CGA GCG GTA AGG GAC AGA CAA ACT GCA-39 Viperin transcription is upregulated by IRF-1 by guest on October 1, 2021 and 59-AAT TCC CAA GCT TGG GTG ATA GGC ACA CAC CTG C-39), We previously established gene expression profiles to uncover the XhoI/HindIII digestion, and cloning in pGL3basic (Promega, Mannheim, Germany). Deletions were introduced into the 21814/+14 viperin-pro- molecular alterations initiated by IRF-1 (24). Among several other moter construct by using the Quick Change Site-Directed Mutagenesis Kit genes with antiviral function, we found the viperin gene to be (Stratagene, La Jolla, CA) according the manufacturer protocol. The se- highly expressed in the presence of functional active IRF-1. This quences of primers are shown in Supplemental Table I. For construction of could be confirmed by transfection of a luciferase-based viperin the viperin expression plasmid pVBC-3mycvigGN viperin mRNA was promoter reporter in combination with an IRF-1 expression vector amplified from NIH3T3 cells (59-AAT TGA ATT CGG GAT GCT GGT GCC CAC TGC-39 and 59-AAT TGT CGA CTC ACC AGT CCA GCT (Fig. 1A). Moreover, IRF-1 M6, a mutant that lacks the DNA TCA GGT CAG C-39), EcoRI/SalI digested, fused to the C terminus of binding domain, was incapable of inducing the promoter activity a myc-tag, and cloned into pVBC-3 in front of an EMCV-derived IRES of the reporter construct (Fig. 1A), indicating that IRF-1 directly r element, eGFP/neo fusion protein cassette. binds to the sequence and thereby activating the viperin promoter. Quantitative real-time PCR To further verify this direct involvement of IRF-1 in the regulation of viperin, we analyzed viperin mRNA level in cells stably ex- Total RNAwas extracted by using the RNeasy kit (Qiagen, Hilden, Germany) and reversely transcribed with Ready-To-Go You-Prime First-Strand Beads pressing an estradiol-activatable IRF-1hER fusion protein (19). (Amersham Biosciences, Buckinghamshire, England). Quantitative RT-PCR Activation of the IRF-1hER fusion protein in the absence and analysis was performed by using a LightCycler (Roche, Mannheim, Ger- presence of the translation inhibitor CHX strongly induced viperin many) and the Quantitect SYBR Green PCR Kit (Qiagen). The data are mRNA synthesis (Fig. 1B,1C). However, the presence of CHX represented as ratios relative to the results of a-actin. Standard curve analysis prevented IFN-g mediated upregulation of viperin supporting the was performed for relative quantification. The following oligonucleotide primers were used: viperin, 59-CTT CAA CGT GGA CGA AGA CA-39 and hypothesis that under this condition IRF-1 gene expression must 59-GAC GCT CCA AGA ATG TTT CA-39; IRF-1, 59-CTC ACC AGG AAC be induced to stimulate viperin transcription (Fig. 1C). Thus, ac- CAG AGG AA-39and 59-TGA GTG GTG TAA CTG CTG TGG-39; b-actin, tivation of viperin transcription by sufficiently expressed IRF-1 is 59-TGG AAT CCT GTG GCATCC ATG AAA C-39 and 59-TAA AAC GCA mediated directly and does not require de novo synthesis of pro- GCT CAG TAA CAG TCC G-39; VSV-Indiana, 59-GGT GGT TAT TCC ATT TTT CG-39and 59-GGT GTT GCA GAC TAT GTT GGA C-39; IRF-7, teins, enhancement of viperin expression can be mediated by el- 59-GAA GAC CCT GAT CCT GGT GA-39and 59-CCA GGT CCA TGA evated IRF-1 levels. GGA AGT GT-39; IFN-b,59-CAC AGG CCATGA AGG AAG AT-39and 59- Bioinformatic promoter analyses of the murine viperin gene TTG GAG CTG GAG CTG CTT AT-39. revealed several consensus response elements for NFkB, AP-1, Luciferase reporter assay NFAT, SP1, and three putative IRF elements (IRF-Es) (Fig. 2A). To determine the binding capacity of IRF-1 to these IRF-E se- Viperin promoter constructs, murine IRF-1 expression plasmids pMT7-IRF- quences EMSAs were performed with in vitro translated IRF-1. 1 (20) or pMT-M6 (20) were cotransfected with pBCRluc (21) using Metafectene (Biontex). Cell lysates were prepared 48 h after transfection. As expected, the control oligonucleotide (IFN stimuated regula- Activities of Firefly and Renilla luciferase were determined using the Dual tory element [ISRE]) was efficiently bound by the IRF-1 protein The Journal of Immunology 5181 Downloaded from FIGURE 1. Viperin is an IRF-1 regulated gene. A, NIH3T3 cells were transiently transfected with a plasmid containing the Firefly luciferase gene driven by the wt viperin promoter (21814/+14) and an expression vector coding for IRF-1 or IRF-1 M6, a mutant that lacks the DNA binding domain. Transfection efficiency was normalized by cotransfection of a constitutive Renilla luciferase expression plasmid. B, NIH3T3 cells, stably expressing an FIGURE 2. IRF-1 directly binds to two proximal IRF-Es of the viperin IRF-1hER fusion protein (NIH3T3IH), were treated with 1 mM b-estradiol to promoter and thereby increases the promoter activity. A, Schematic map of http://www.jimmunol.org/ activate IRF-1. Relative viperin mRNA concentration was measured by the three IRF-1 binding sites (IRF-E-1, -2, and -3) in the 2000 bp upstream quantitative real-time PCR at the indicated times. C, NIH3T3IH cells were region of the murine viperin promoter. Their position in the promoter is pretreated with 60 mg/ml CHX, 2 h before b-estradiol–induced activation of represented with respect to the transcriptional start. B, Formation of IRF-1/ the IRF-1hER fusion protein (IRF-1 and CHX) or IFN-g (100 U/ml) treat- DNA complexes was tested by EMSA using the sequence of the indicated ment (IFN-g and CHX). All data represent mean values with SD from at IRF-E sites and in vitro translated IRF-1. A probe with the ISRE sequence least two independent experiments. of the IFN-b promoter was used as positive control. C, EMSA was per- formed as in B but with oligonucleotides representing mutated IRF-E se- quences. D, NIH3T3IH cells transiently transfected with a Renilla (Fig. 2B). In addition, complex formation between IRF-1 and the

luciferase reporter plasmid (pBCRluc), a plasmid with the Firefly lucif- by guest on October 1, 2021 oligonucleotides representing IRF-E-3, IRF-E-2, and IRF-E-2/3 erase gene driven by the wt viperin promoter (21814/+14) or reporter has been detected (Fig. 2B). The protein/oligonucleotide binding constructs containing mutations in one of the IRF-E sites. Activation of could be prevented by introducing deletions into the IRF-E core IRF-1 by b-estradiol (1 mM) treatment was performed 24 h before har- sequence (Fig. 2C), showing the sequence specificity of these vesting cells for the dual luciferase assay. Relative luciferase activities of interactions. Consistent with these observations, the transiently Firefly luciferase were calculated by normalization to Renilla luciferase transfected viperin promoter reporter containing the deleted IRF- activity. The graph shows the fold increase of relative luciferase activities in cells treated with b-estradiol (IRF-1) compared with untreated cells (C). E-3 showed significantly lower activation by IRF-1, whereas al- teration of the IRF-E-2 sequence led to a complete loss of reporter gene expression (Fig. 2D). gistically interacts to induce IRF-1 transcription (26). Therefore, we examined whether STAT1 also plays a role in the IRF-1– IRF-1 is essential for IFN-g and VSV induced viperin mediated viperin induction on virus infection. First of all, we expression investigated whether STAT1 itself or synergistically with IRF-1 Viperin is highly induced on IFN stimulation and by infection with influences the viperin promoter activity. Transfection of NIH3T3 a broad range of viruses. To investigate whether IRF-1 exerts in- cells with an IRF-1 expression vector induced the viperin pro- fluence on these events, we tested wild-type (wt) and IRF-1–de- moter activity in a dose-dependent manner, whereas expression of ficient (IRF-12/2) MEFs for alterations in viperin mRNA levels STAT1 had no significant influence, both in the absence or caused by IFN-b, IFN-g, VSV, and NDV, respectively. As ex- presence of ectopic IRF-1 (Fig. 4A). Therefore, a direct influence pected, in wt MEFs, increased viperin mRNA levels could be of STAT1 on the viperin promoter activity is unlikely. In addition, detected as a consequence of treatment with all four stimuli (Fig. performed quantitative RT-PCR analyses of STAT12/2 MEFs 3A). Stimulation of IRF-12/2 cells with IFN-b or infection with showed only weak induction of viperin by NDV but no induction NDV leads to the induction of viperin (Fig. 3B) indicating that after VSV infection (Fig. 4B). In contrast, the transient expression IRF-1 plays no role under these conditions. In contrast, IFN-g of IRF-1 significantly enhanced the amount of viperin mRNA in failed to induce viperin expression in IRF-12/2 cells, suggesting STAT1-deficient MEFs (Fig. 4C). This supports our hypothesis an important role of IRF-1 in IFN-g mediated viperin induction. A that an increase of cellular IRF-1 expression is sufficient to ac- more revealing finding was that in the absence of the IRF-1 gene tivate the viperin promoter and that STAT1 is essential for stim- activity, the VSV-mediated viperin induction failed (Fig. 3B), in- ulation of IRF-1 transcription. These results also indicate dicating that the presence of the IRF-1 is essential to activate the a STAT1-dependent mechanism of viperin induction during NDV viperin promoter under these conditions. infection, that results from the involvement of STAT1 in other IFN-g is known to be a potent IRF-1 inducer (25). Its binding IRF-1 independent antiviral signaling pathways, namely, the type to the IFN-g receptor activates STAT1 and NFkB, which syner- I IFN pathway. 5182 IFN INDEPENDENT, IRF-1 DEPENDENT VIPERIN INDUCTION Downloaded from

FIGURE 3. IFN-g and VSV mediated enhancement of viperin expression is impaired in IRF-1 deficient MEFs. A, The wt and (B) IRF-12/2 cells were FIGURE 4. Involvement of STAT1 in viperin induction. A, NIH3T3 cells treated with IFN-b (500 U/ml) or IFN-g (100 U/ml) or infected with VSVor were transfected with a viperin reporter plasmid and increasing amounts of NDV for 1 h. Cells were harvested at the indicated time points and analyzed IRF-1 or STAT1 (0, 0.125, 0.25, and 0.5 mg). Transfection efficiency was for their relative viperin mRNA amounts by quantitative real-time PCR. All normalized by cotransfection of a constitutive Renilla luciferase expression http://www.jimmunol.org/ plasmid. Relative luciferase values were shown normalized to activities data represent mean values with SD from at least two independent experi- 2/2 ments. B, pp , 0.05; ppp , 0.01; pppp , 0.001, Student t test. obtained without IRF-1 or STAT1 cotransfection. B,STAT1 cells were infected with VSVor NDV for 1 h and harvested 4, 8, or 24 h postinfection. Viperin mRNA was determined by quantitative RT-PCR and normalized to An intact IFN system is dispensable for the enhanced expression a-actin. C, Quantitative real-time PCR analysis of viperin expression in and the antiviral function of viperin in VSV infected cells STAT12/2 MEFs 24 h after transient transfection of an IRF-1 expression Boudinot et al. showed that viperin expression in a dendritic cell line plasmid (IRF-1) or an empty control vector (2). All data represent mean infected with VSV is upregulated even in the presence of Abs values with SD from at least two independent experiments. neutralizing type I IFN activity, namely, in an IFN-a/b independent 2 2 by guest on October 1, 2021 manner (9). The results presented in Fig. 3 reveal that IRF-1 is es- titer in IFNAR / were higher compared with those in wt cells, sential for IFN-g and VSVinduced viperin expression. To assess the confirming the role of IFN in antiviral defense. Nevertheless, this possible involvement of IRF-1 in an IFN independent regulation of experiment reveals viperin to be very effective in diminishing viperin, type I receptor deficient (IFNAR2/2) MEFs were cultured VSV replication, even in the absence of functional IFN signaling. in the presence of IFN-b, IFN-g, or infected with VSVand NDV,and IRF-1 provides a bypass mechanism of viperin induction analyzed for changes in viperin mRNA concentration. We can ex- during infection with viruses that counteract the IFN system clude an IFN effect dependent on IFN-g, because only T, NKT, and NK cells are able to express IFN-g. The IRF-1–dependent viperin As many other viruses, VSVevolved mechanisms to circumvent the induction by IFN-g and VSV was still detectable in the absence of innate immune defense by blocking the IFN signaling (27). Whereas type I IFN signaling (Fig. 5A). Interestingly, NDV also activated activation of IRF-3 and induction of IFN-b expression were found in viperin expression independent of type I IFN (Fig. 5A). In addition, VSV and AV2 infected cells, IRF-7 mRNA expression was not de- increased IRF-1 mRNA concentrations were detected in VSV in- tectable in VSV infected cells (Supplemental Fig. 2). It seems that fected IFNAR2/2 MEFs, but mRNA levels were only marginally the IRF-1 dependent pathway of viperin induction is of particular but statistically significant changed by NDV infection (Fig. 5B). importance when cells cannot activate the IFNAR response on in- These findings suggest that infected cells can respond to both viruses fection. Conversely, the IRF-1 mediated induction of viperin ex- via IFN independent mechanisms. However, the IRF-1 dependent pression should be dispensable when viruses fail to block the IFN 2/2 pathway is indispensible only in cells infected with VSV. signaling. To test this hypothesis, IRF-1 MEFs were infected To demonstrate the relevance of the VSV-induced IRF-1 de- with a mutant VSV strain (AV2), which lacks the ability to suppress pendent pathway, we examined whether the induction of viperin is IFN gene expression (28). AV2 and NDV, but not wt VSV, infected adequate to counteract viral replication. The wt and IFNAR2/2 cells responded with substantial secretion of IFN (Fig. 6A). Con- MEFs, transiently transfected with an expression vector encoding sistently, an increase of viperin mRNA, comparable to that of NDV for viperin or a control vector, were infected with VSV for 1 h. In infected MEFs (Fig. 3B), was measured on AV2 infection (Fig. 6B), the 24-h postinfection we analyzed viral replication by quantify- showing that the capability of VSV to prevent IFN expression forces ing the concentration of infectious viruses in the supernatant. The the cell to induce viperin via an IFN independent, but IRF-1 de- expression of viperin led to a strong antiviral response against pendent pathway. VSV, in both wt- and IFNAR-deficient cells (Fig. 5C, Supple- mental Fig. 1). The inhibition of viral replication was comparable Discussion to the effect of IFN treatment. Depletion of type I IFN in the A multiplicity of viruses, LPSs and dsRNA induce the viperin supernatant by specific Abs prevented the IFN induced but not the expression through IFN-dependent and IFN-independent mecha- viperin induced inhibition of viral replication. Generally, VSV nisms. The IFN-dependent viperin induction can be mediated by The Journal of Immunology 5183

FIGURE 6. IRF-1 provides a redundant mechanism to bypass the type I IFN system. IRF-12/2 MEFs were infected for 1 h with NDV, VSV (MOI 0.01), or the mutated VSV strain AV2 (MOI 0.01). A, Secreted type I IFN was determined in the cell supernatant 24 h postinfection by an antiviral assay. B, At the indicated time periods after AV2 infection, total RNA was extracted, reversely transcribed, and analyzed for viperin mRNA amount by quantitative real-time PCR. All data represent the mean value with SD of at least two independent experiments. MOI, multiplicity of infection.

a consequence of viperin-induced disruption of lipid-raft micro- Downloaded from domains, which results from the interaction and inactivation of the enzyme farnesyl diphospate synthease with viperin (11). VSV does not bud from lipid rafts, which is indicative for the presence of other antiviral mechanisms. Our data show, that viperin expression also inhibits the replication of VSV more than 1000-fold (Fig. 5C). This strong inhibition of virus replication by an intrinsic effect is http://www.jimmunol.org/ only comprehensible in the unlikely case of 100% transfection efficiency. Transfection efficiency was determined by the cotransfer of a GFP expression vector and is 60% or higher (data not shown). This transfection efficiency is not sufficient for a 1000-fold re- duction of viral replication. One possible explanation is that a very low expression level of viperin that might be induced by a trans- FIGURE 5. The increase in viperin transcription during VSV infection is IRF-1 dependent and type I IFN independent. Relative (A) viperin and (B) fection of nearly 100% of cells is sufficient to induce an antiviral response. Alternatively, paracrine effects could be responsible for IRF-1 mRNA amounts normalized to a-actin were determined by quanti- by guest on October 1, 2021 tative real-time PCR in IFNAR2/2 MEFs treated with IFN-b (500 U/ml), this phenomenon. We checked the supernatant for further antiviral IFN-g (100 U/ml), VSV, or NDV at indicated times. B, pp , 0.05; ppp , effects but could not detect a respective activity (data not shown). 0.01; pppp , 0.001, Student t test. C, The wt and IFNAR2/2 cells were Previous studies with IRF-1 deficient MEFs demonstrated an in- transiently transfected with an empty vector (mock) or a viperin expression volvement of IRF-1 in the type I IFN-dependent antiviral response vector and infected with VSV (MOI 0.01) for 1 h. In addition, cells were against ECMV infection (29) and in an IFN-g mediated mechanism treated with 500 U/ml IFN-b and/or Abs against 2000 U/ml type I IFN. Viral preventing vaccinia virus replication (30). In addition, it was found titers (PFU/ml) were determined 24 h postinfection by a plaque formation that IRF-1 exerts a direct antiviral response, which is consistent with assay. All data represent the mean value with SD of at least two independent experiments. MOI, multiplicity of infection. type I IFNs, IFN-g, and type III IFNs. Previous publications showed that activation of the type I IFN signaling pathway en- hances the viperin transcription through the STAT1/STAT2/IRF-9 complex termed ISG factor 3 (ISGF3) by binding to the promoter response element ISRE (8, 12). In this article, we reveal IRF-1 as a mediator for the VSV and IFN-g amplified viperin expression (Fig. 3). IRF-1 mediates its activity by binding to conserved IRF elements in the promoter of its target genes. Our data demonstrate that the viperin promoter contains two IRF-E sites, which are necessary for IRF-1 mediated promoter activation (Fig. 2). This FIGURE 7. Schematic presentation of viperin induction. Virus infection overlap of gene induction by IRF-1 and type I IFNs is not as- could lead to the induction of the type I IFN pathway. The secreted IFN acts tonishing, because the IRF binding sites and the one from IFN- in an autocrine and paracrine manner, leading to the activation of the het- induced ISGF3 complex are very similar. erotrimeric STAT1/STAT2/IRF-9 complex ISGF3. By its function as a tran- Viperin limits replication of viruses like HCMV (10), VSV- scriptional activator ISGF3 mediates the induction of ISGs, like viperin. Independently, virus infection can lead to the transcriptional induction of pseudotyped HIV (14), and HCV (15). The exact mechanism by IRF-1, which directly activates the viperin promoter. Viruses, like NDV, can which viperin limits the viral replication is unknown. In HCMV activate both pathways on infection, which leads to viperin induction in the infected cells the expression of viperin inhibits the expression of absence of IFNAR or IRF-1. Many viruses, like VSV, developed strategies to several structural proteins critical for viral assembly and maturation block the expression or function of type I IFN. VSV infection inhibits the (10). In contrast, in influenza A virus infected cells, viperin inhibits function of type I IFNs but activates the IRF-1 pathway that enables the the budding of the virus from the plasma membrane. This is infected cells to induce an antiviral response. 5184 IFN INDEPENDENT, IRF-1 DEPENDENT VIPERIN INDUCTION

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