Communication

Antiviral Activity of Porcine Interferon Regulatory Factor 1 against Swine Viruses in Cell Culture Yongtao Li, Hongtao Chang, Xia Yang, Yongxiang Zhao, Lu Chen, Xinwei Wang, Hongying Liu, Chuanqing Wang and Jun Zhao * Received: 7 September 2015 ; Accepted: 3 November 2015 ; Published: 17 November 2015 Academic Editor: Curt Hagedorn College of Animal Husbandry & Veterinary Science, Henan Agricultural University, Zhengzhou 450002, China; [email protected] (Y.L.); [email protected] (H.C.); [email protected] (X.Y.); [email protected] (Y.Z.); [email protected] (L.C.); [email protected] (X.W.); [email protected] (H.L.); [email protected] (C.W.) * Correspondence: [email protected]; Tel.: +86-0371-6355-5340; Fax: +86-0371-6355-8529

Abstract: Interferon regulatory factor 1 (IRF1), as an important transcription factor, is abundantly induced upon virus infections and participates in host antiviral immune responses. However, the roles of porcine IRF1 (poIRF1) in host antiviral defense remain poorly understood. In this study, we determined that poIRF1 was upregulated upon infection with viruses and distributed in nucleus in porcine PK-15 cells. Subsequently, we tested the antiviral activities of poIRF1 against several swine viruses in cells. Overexpression of poIRF1 can efficiently suppress the replication of viruses, and knockdown of poIRF1 promotes moderately viral replication. Interestingly, overexpression of poIRF1 enhances dsRNA-induced IFN-β and IFN-stimulated response element (ISRE) promoter activation, whereas knockdown of poIRF1 cannot significantly affect the activation of IFN-β promoter induced by RNA viruses. This study suggests that poIRF1 plays a significant role in cellular antiviral response against swine viruses, but might be dispensable for IFN-β induction triggered by RNA viruses in PK-15 cells. Given these results, poIRF1 plays potential roles in cellular antiviral responses against swine viruses. Keywords: porcine IRF1; antiviral activity; IFN-β; IFN-stimulated genes

1. Introduction Virus infection and subsequent replication poses a significant threat to the host immune system. As a result, every living species has evolved mechanisms for recognizing viruses and counteracting them through immune responses. Type I interferons (IFN) are the central components of early innate immune response against viral infection. Generally, when a virus is invading a cell, cellular pattern recognition receptors (PRRs) can recognize viral nucleic acids, and transmit signals to downstream adaptors, which eventually results in the activation of transcription factors of IFN regulatory factor (IRF) family, primarily including IRF3 and/or IRF7, to induce the expression of type I IFN [1]. Type I IFN bind a heterodimeric transmembrane receptor termed the IFNα receptor (IFNAR) to activate interferon-stimulated gene factor 3 (ISGF3) via the JAK-STAT signaling pathway and induce the coordinated upregulation of hundreds of interferon-stimulated genes (ISGs) that orchestrate an antiviral state in the cells [2]. In addition to IRF3/7, IRF1 also plays a significant role in type I IFN induction in cell-specific fashions. As the first member of IRF family, IRF1 is constitutively expressed in most cell types and is dramatically induced upon viral infection, treatment with dsRNA or IFN stimulation [3–5]. Early research has indicated that IRF1, as an important mediator, regulates the expression of type I IFN [4,6,7]. Subsequently, analysis of IRF1´{´ cells from mice has shed light on the unnecessary role of IRF1 in IFN-β induction by virus infection [8–10]. Although, whether IRF1 is directly involved in IFN Viruses 2015, 7, 5908–5918; doi:10.3390/v7112913

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in IFN-β induction by virus infection [8–10]. Although, whether IRF1 is directly involved in IFN controversial, increasing studies show that IRF1 plays a pivotal role in host induction remains controversial, [11–15]. Recently, systematic screening of antiviral antiviral responses against a wide set of viruses [11–15]. effectors has identified human IRF1 as a broadly acting effector effector to to defend defend against against diverse diverse viruses viruses effectors has identified human IRF1 as a broadly acting tested, highlighting highlighting the the physical physical relevance relevance of of IRF1 IRF1 in in the the innate innate antiviral antiviral responses responses[16,17]. [16,17]. tested, In our our previous previous studies, studies, transcriptome transcriptome profiling profiling analyses analyses showed showed that that ISGs ISGs were were the the most most highly highly In induced genes in pig lungs after H1N1 influenza virus infection at an early stage [18]. We investigated induced genes in pig lungs after H1N1 influenza virus infection at an early stage [18]. We investigated the antiviral antiviral activity activity of of porcine porcine ISGs ISGs and and initially initially identified identified porcine porcine IRF1 IRF1 (poIRF1) (poIRF1) as as the the most most active active the effector in the defense against influenza virus. Although many studies have already focused on effector in the defense against influenza virus. Although many studies have already focused on antiviral functions functions of of human human or or mouse mouse IRF1, IRF1, the the antiviral antiviral functions functions of of poIRF1 poIRF1 are are poorly poorly understood. understood. antiviral Therefore, we we sought sought to to investigate investigate the the antiviral antiviral activity activity of of poIRF1 poIRF1 against against four four swine swine viruses viruses in in Therefore, cultured porcine kidney epithelial cells (PK-15) and investigated the possible mechanism by which cultured porcine kidney epithelial cells (PK-15) and investigated the possible mechanism by which IRF1 exerts exerts its its antiviral antiviral effects. effects. This role in in cellular cellular IRF1 This study study suggests suggests that that poIRF1 poIRF1 plays plays aa significant significant role antiviral response response against against swine swine viruses, viruses, but but might might be be dispensable dispensable for for IFN-β IFN-β induction induction triggered triggered by by antiviral RNA viruses in PK-15 cells. RNA viruses in PK-15 cells. 2. Results Results Firstly, Firstly, the the subcellular subcellular localization localization of poIRF1 protein protein was investigated in PK-15 cells upon poly(I:C) or SIV infection. As shown in Figure 1A, poIRF1 (in red) accumulated primarily in nucleus of both poly(I:C)- and SIV-stimulated SIV-stimulated cells. cells. To determine the expression patterns of poIRF1, PK-15 cells were stimulated with poly(I:C) or infected with swine influenza virus (SIV), vesicular stomatitis virus (VSV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), and poIRF1 transcripts were measured measured by by RT-qPCR RT-qPCR at at various various time time points. points. Our data showed that IRF1 mRNA was expressed at a low basal level in PK-15 cells, and was induced rapidly after poly(I:C) treatment at very considerable levels at 6 hours post infection (hpi) and reached to the peak at 24 hpi (72-fold versus control) continuously and remained high at control) (Figure (Figure1B). 1B).In InSIV-infected SIV-infectedcells, cells,poIRF1 poIRF1increased increased continuously and remained high 36 hpihpi (45-fold vs.vs. control) (Figure 1C). at 36 (45-fold control) (Figure 1C). In TGEV-infected TGEV-infected cells, cells, poIRF1 poIRF1increased increasedcontinuously continuouslyand and reached peak at hpi 24 (11hpi reached to to thethe peak at 24 (11-fold versus control) (Figure 1D). However, poIRF1 exhibited slight increase in response fold versus control) (Figure 1D). However, poIRF1 exhibited only aonly slighta increase in response to VSV to VSV infection, the expression highest expression levels (4.5-fold vs. at control) 24 hpi1E). (Figure 1E). In infection, with thewith highest levels (4.5-fold vs. control) 24 hpi at (Figure In addition, addition, poIRF1 expression level exhibited large differences in the PRV-QXX virulent PRV-QXX (not significant poIRF1 expression level exhibited large differences in the virulent (not significant change) and attenuated PRV-BaPRV-Ba (10-fold(10-fold vs. control at 24 hpi) cells cells (Figure 1F,G), indicating the change) and attenuated vs. control at 24infected hpi) infected (Figure 1F,G), indicating possible relationship between viral virulence and the possible relationship between viral virulence andthe theantagonistic antagonisticcapability capabilityofof virus virus to to IRF1 expression. In In brief, brief, the the expression expression patterns patterns of of poIRF1 induced by different viruses in PK-15 cells exert different different antiviral antiviral activities activities against against these these swine swineviruses. viruses. suggested that poIRF1 might exert

Figure 1. 1. Cont. Cont. Figure

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Figure 1. 1.poIRF1 induced PK-15 dsRNA stimulation viral infections. (A) Figure poIRF1 isisinduced in in PK-15 cellscells uponupon dsRNA stimulation and viraland infections. (A) Detection Detection of poIRF1 and influenza virus Hemagglutinin (HA) protein by immunofluorescence of poIRF1 and influenza virus Hemagglutinin (HA) protein by immunofluorescence microscopy. PKmicroscopy. cellswere on coverslips treated by poly(I:C) or12 SIV infection. At 12 15 cells onPK-15 coverslips treated bywere poly(I:C) stimulation or SIVstimulation infection. At h post-treatment, h post-treatment, IFA was performed for visualization of the distribution of poIRF1 and HA IFA was performed for visualization of the distribution of poIRF1 and HA protein detected byprotein Cy3detected by Cy3- and FITC-labeled secondary antibodies respectively of and visualization ofby cell nuclei and FITC-labeled secondary antibodies respectively and visualization cell nuclei stained DAPI. stained by DAPI. Confocal images were on a LSM510 METAscale microscope; scale(B–G) bar, 50The µm; Confocal images were captured on a captured LSM510 META microscope; bar, 50 µm; (B–G) The expression patterns in PK-15 cells with stimulated with poly(I:C) or viruses; infectedPKwith expression patterns of poIRF1 of in poIRF1 PK-15 cells stimulated poly(I:C) or infected with viruses; PK-15 were stimulated with (B) or infected with SIV VSV (E) 15 cells were cells stimulated with poly(I:C) (B)poly(I:C) or infected with SIV (C); TGEV (D); (C); VSVTGEV (E) and(D); PRV-QXX and(F)PRV-QXX (F) (G). and At PRV-Ba (G). At times, the indicated times, theofquantification of poIRF1 transcripts and PRV-Ba the indicated the quantification poIRF1 transcripts was performed RT-qPCR at 12, 24 and Relative values of poIRF1 gene were normalized to wasbyperformed by6,RT-qPCR at36 6, hpi. 12, 24 and 36quantitative hpi. Relative quantitative values of poIRF1 gene were the level of gene expression between infected and control cellsand werecontrol analyzed normalized toGAPDH. the levelDifferences of GAPDH.ofDifferences of gene expression between infected cells byanalyzed using the by Student’s t-test. The results were graph formatinasgraph mean format ± SD. as mean ˘ SD. were using the Student’s t-test. Theplotted resultsinwere plotted

In order to determine the effect of poIRF1 overexpression on virus replication, PK-15 cells stably In order to determine the effect of poIRF1 overexpression on virus replication, PK-15 cells stably expressing poIRF1 or mock cells were established by PB transposon systems, designed as PK-15/IRF1 expressing poIRF1 or mock cells were established by PB transposon systems, designed as PK-15/IRF1 and PK-15/PB respectively (Figure 2A). Western blot results indicated high level expression of poIRF1 and PK-15/PB respectively (Figure 2A). Western blot results indicated high level expression of poIRF1 in PK-15/IRF1 cells relative to PK-15/PB and normal PK-15 cells (Figure 2B). Then, PK-15/PB and PKin 15/IRF1 PK-15/IRF1 cells infected relative with to PK-15/PB andatnormal PK-15 of cells (Figure 2B).ofThen, PK-15/PB cells were swine viruses a multiplicity infection (MOI) 0.1 TCID 50/mL andforPK-15/IRF1 cells were infected with swine viruses at a multiplicity of infection (MOI) SIV and TGEV, at a MOI of 0.1 PFU/mL for VSV and PRV-QXX. After infections, viral titers inof the0.1 TCID /mL for SIV and TGEV, at a MOI of 0.1 PFU/mL for VSV and PRV-QXX. After infections, 50 culture supernatants were determined at the indicated time points. The results showed that the SIV viral titers in PK-15/IRF1 the culturecells supernatants werebydetermined at thecompared indicatedwith time points. results titers from were reduced 14- and 100-fold these from The PK-15/PB showed that titers from PK-15/IRF1 cellsfrom werepoIRF1-overexpressed reduced by 14- and 100-fold compared cells at 24 the and SIV 48 hpi, respectively. TGEV titers cells were reduced with by these from PK-15/PB cells at 24 and 48 hpi, respectively. TGEV titers from poIRF1-overexpressed approximately 63-fold and 126-fold compared with those in PK-15/PB cells at 24 and 48 hpi, cells were reduced by approximately 63-fold and cells 126-fold with thosereadily in PK-15/PB cells respectively. Moreover, detection of VSV-infected undercompared microscope revealed detectable cytopathic effects (CPE) at 12Moreover, and 24 hpidetection in PK-15/PB, but rarely detectable CPE microscope in PK-15/IRF1 cells at 24 and 48 hpi, respectively. of VSV-infected cells under revealed (Figure not shown). In accordance with microscope detection, VSV titersbut in the celldetectable supernatants readily detectable cytopathic effects (CPE) at 12 and 24 hpi in PK-15/PB, rarely CPE revealed up to 630-(Figure and 10,000-fold reduction in PK-15/IRF1 cells compared with these in titers PK-15/PB in PK-15/IRF1 cells not shown). In accordance with microscope detection, VSV in the at 12 and revealed 24 hpi. We tested inhibition of a dsDNA virus, PRV, and cellcells supernatants up also to 630andpoIRF1-mediated 10,000-fold reduction in PK-15/IRF1 cells compared with detected obviously reduced CPE in PK-15/IRF1 cells compared with PK-15/PB cells. Plaque counts these in PK-15/PB cells at 12 and 24 hpi. We also tested poIRF1-mediated inhibition of a dsDNA virus, revealed markedly a 7- and 5-fold reduction of PRV titers incompared PK-15/IRF1 cells comparedcells. withPlaque PKPRV, and detected obviously reduced CPE in PK-15/IRF1 cells with PK-15/PB 15/PB cells (Figure 2C). The results indicated that overexpression of poIRF1 potently inhibits virus counts revealed markedly a 7- and 5-fold reduction of PRV titers in PK-15/IRF1 cells compared with replication. PK-15/PB cells (Figure 2C). The results indicated that overexpression of poIRF1 potently inhibits To determine whether endogenous poIRF1 expression affects viral replication, siRNA targeting virus replication. to poIRF1 (si-IRF1) was transfected into PK-15 cells with a non-targeting siRNA as control (si-Ctrl). To determine whether endogenous poIRF1 expression affects viral replication, siRNA targeting The knockdown efficiency of poIRF1 was determined by RT-qPCR. The results showed that si-IRF1 to poIRF1 (si-IRF1) was transfected into PK-15 cells with a non-targeting siRNA as control (si-Ctrl). transfection led to a 50%, 77% and 38% decrease in IRF1 transcription at 24, 36 and 48 h post Thetransfection, knockdownrespectively, efficiency of poIRF1 was by3A). RT-qPCR. The protein results has showed that si-IRF1 compared with determined si-Ctrl (Figure As the IRF1 a short half-life, transfection ledlevels to a most 50%,probably 77% and 38% decrease inprotein IRF1 transcription at Therefore, 24, 36 and h post IRF1 mRNA correlate with IRF1 abundance [19]. we48 further transfection, respectively, compared with si-Ctrl (Figure 3A). As the IRF1 protein has a short half-life, detected the knockdown effect of poIRF1 protein levels at 36 h post transfection and found that siIRF1 mRNA levels most probably correlate with IRF1ofprotein [19]. Therefore, IRF1 transfection resulted to a significant decrease poIRF1abundance protein in PK-15, PK-15/PB we andfurther PKdetected the knockdown of poIRF1 protein 36 hcells postwere transfection andby found thatand si-IRF1 15/IRF1 cells comparedeffect with si-Ctrl (Figure 3B). levels Then, at PK-15 transfected si-IRF1 sitransfection resulted to a significant decrease of poIRF1 protein in PK-15, PK-15/PB and PK-15/IRF1 3 cells compared with si-Ctrl (Figure 3B). Then, PK-15 cells were transfected by si-IRF1 and si-Ctrl for

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36 and36then infected abovewith swine viruses for determining titers in the culture Ctrlh, for h, and thenwith infected above swine viruses for determining titers insupernatants the culture at 24 hpi. The results showed that knockdown of poIRF1 approximately led to a 2.8-, 4.0-, and supernatants at 24 hpi. The results showed that knockdown of poIRF1 approximately led to10a 2.8-, 2.7-fold increase in viral growth for SIV, TGEV, VSV TGEV, and PRV-QXX, (Figure 3C).(Figure Taken 4.0-, 10- and 2.7-fold increase in viral growth for SIV, VSV and respectively PRV-QXX, respectively together, our data suggest that knockdown of IRF1 enhanced moderately replication of SIV, TGEV, 3C). Taken together, our data suggest that knockdown of IRF1 enhanced moderately replication of VSV and PRV in PK-15 cells. SIV, TGEV, VSV and PRV in PK-15 cells. Viruses 2015, 7, page–page

Ctrl for 36 h, and then infected with above swine viruses for determining titers in the culture supernatants at 24 hpi. The results showed that knockdown of poIRF1 approximately led to a 2.8-, 4.0-, 10- and 2.7-fold increase in viral growth for SIV, TGEV, VSV and PRV-QXX, respectively (Figure 3C). Taken together, our data suggest that knockdown of IRF1 enhanced moderately replication of SIV, TGEV, VSV and PRV in PK-15 cells.

Figure 2. 2. Overexpression virus replication. replication. (A) Overexpression of of poIRF1 poIRF1 potently inhibits virus (A) PK-15 PK-15 cells stably were established established by by PB transposon transposon systems. After puromycin treatment, the single expressing poIRF1 were puromycin-resistant and GFP-positive cell clones, designated asPK-15/IRF1 PK-15/IRF1 and PK-15/PB, puromycin-resistant and GFP-positive cell clones, designated as and were Figure 2. Overexpression of poIRF1 potently inhibits virus replication. (A) PK-15 cellsPK-15/PB, stably bar, 100 µm; protein by Western expressing poIRF1 were established by PB transposon systems. puromycin treatment, the single captured on a fluorescence microscope; scale bar, (B)After Detection of poIRF1 puromycin-resistant and GFP-positive celland clones, designated were as PK-15/IRF1 and PK-15/PB, were blot. Protein lysates from PK-15, PK-15/PB PK-15/IRF1 denatured in buffer. PK-15/PB and PK-15/IRF1 were denatured in SDS SDS sample captured on a fluorescence microscope; scale bar, 100 µm; (B) Detection of poIRF1 protein by Western SDS-PAGE followed by Western Western blot blot using antibodies detecting Denatured lysates were analyzed by SDS-PAGE blot. Protein lysates from PK-15, PK-15/PB and PK-15/IRF1 were denatured in SDS sample buffer. was used as a loading control; (C) The impact of poIRF1 poIRF1, asDenatured indicated. β-actin overexpression lysates were analyzed by SDS-PAGE followed by Western blot using antibodies detecting replication. PK-15 β-actin cells stably expressing orThe with on viral replication. vector aloneoverexpression were incubated with poIRF1, as indicated. was used as a loadingpoIRF1 control; (C) impact of poIRF1 on viral replication. PK-15 stablysupernatants expressing poIRF1 or with vector alone indicated viruses for 24 h and 48 cells h before were harvested. Thewere viralincubated titers inwith supernatants were harvested. indicated viruses for 24 h and 48 h before supernatants were harvested. The viral titers in supernatants were measured measured with with TCID TCID50 50 assay or plaque assay. were measured with TCID50 assay or plaque assay.

Figure 3. Cont.

Figure 3. Cont. 4

Figure 3. Cont.

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Figure Knockdown of of poIRF1 poIRF1 promotes promotes moderately moderately virus Figure 3. 3.Knockdown virus replication. replication. (A) (A)The Theknockdown knockdown efficiency of poIRF1 was determined by RT-qPCR. PK-15 cells were transfected with si-IRF1 oror si-Ctrl efficiency of poIRF1 was determined by RT-qPCR. PK-15 cells were transfected with si-IRF1 si-Ctrl Figure 3. Knockdown of poIRF1 promotes moderately virus replication. (A) The knockdown for 24 h, 36 h and 48 h, and subsequently the poIRF1 expression in transfected cells was verified at forefficiency 24 h, 36 h and 48 h, and subsequently the poIRF1 expression in transfected cells was verified of poIRF1 was determined by RT-qPCR. PK-15 cells were transfected with si-IRF1 or si-Ctrl at mRNA levels by RT-qPCR; (B) The knockdown efficiency of poIRF1 was checked by Western blot. mRNA RT-qPCR; The knockdown efficiency of poIRF1 was checked by Western for 24levels h, 36 hbyand 48 h, and(B) subsequently the poIRF1 expression in transfected cells was verified blot. at PK-15, PK-15/PB and PK-15/IRF1 cells were transfected with si-IRF1 or si-Ctrl for 36 h. The poIRF1 PK-15, PK-15/PB PK-15/IRF1 cells were transfected with si-IRF1was or si-Ctrl forby 36Western h. The poIRF1 mRNA levels byand RT-qPCR; (B) The knockdown efficiency of poIRF1 checked blot. expression in transfected cells was verified at protein levels by Western blot assay. β-actin was used expression in transfected cells was cells verified protein levels Western blot assay. used PK-15, PK-15/PB and PK-15/IRF1 wereattransfected withby si-IRF1 or si-Ctrl for 36β-actin h. The was poIRF1 as a loading control; (C) The effect of knockdown of poIRF1 on viral replication. PK-15 cells were as expression a loading control; (C) The of knockdown of levels poIRF1 viral replication. PK-15was cells were in transfected cellseffect was verified at protein by on Western blot assay. β-actin used transfected with si-IRF1 or si-Ctrl for 36 h. Then, cells were incubated with indicated viruses for 24 h transfected withcontrol; si-IRF1(C) or The si-Ctrl for of 36 knockdown h. Then, cells incubated indicated viruses 24 h as a loading effect of were poIRF1 on viral with replication. PK-15 cells for were before supernatants were harvested. The viral titers in supernatants were measured with TCID50 assay before supernatants were or harvested. The in supernatants were measured with transfected with si-IRF1 si-Ctrl for 36 h. viral Then, titers cells were incubated with indicated viruses forTCID 24 h 50 or plaque assay. before supernatants assay or plaque assay.were harvested. The viral titers in supernatants were measured with TCID50 assay or plaque assay. of IFN response in virus-infected cells is an event central to innate antiviral The induction

immunity. To determine poIRF1 could regulatecells IFN-β PK-15 cells, we detected The induction of IFNwhether response in virus-infected is response an eventincentral to innate antiviral The induction of IFN response in virus-infected cells is an event central to innate antiviral the contribution of poIRF1 to the induction IFN-β and ISGs. As shown in Figure immunity. To determine whether poIRF1 couldofregulate IFN-β response in PK-15 cells,4A, wepoIRF1 detected immunity. To determine whether poIRF1 could regulate IFN-β response in PK-15 cells, we detected can result to in the significant activation of IFN-β promoter (4.5-fold) and upregulated theoverexpression contribution of poIRF1 induction of IFN-β and ISGs. As shown in Figure 4A, poIRF1 the contribution of poIRF1 to the induction of IFN-β and ISGs. As shown in Figure 4A, poIRF1 expression of can IFN-β transcript (8.4-fold)activation relative toofthat in control cells. Upon stimulation with overexpression result in significant IFN-β promoter (4.5-fold) and upregulated overexpression can result in significant activation of IFN-β promoter (4.5-fold) and upregulated poly(I:C), cells overexpressing poIRF1 produced significantly higher levels of IFN-β promoter expression of ofIFN-β cells. Upon Uponstimulation stimulation with expression IFN-βtranscript transcript(8.4-fold) (8.4-fold) relative relative to to that that in in control control cells. with activity cells and overexpressing IFN-β mRNA poIRF1 than control cells. Meanwhile,higher we found that poIRF1 couldactivity also poly(I:C), produced significantly levels of IFN-β promoter poly(I:C), cells overexpressing poIRF1 produced significantly higher levels of IFN-β promoter significantly enhance ISRE activation relative to that in control cells, and when followed by and IFN-β and mRNA than control cells. Meanwhile, we found thatfound poIRF1 could also significantly activity IFN-β mRNA than control cells. Meanwhile, we that poIRF1 could also transfection of poly(I:C), ISRE activation was further promoted to a higher level (Figure 4A). On the enhance ISRE activation relative to thatrelative in control cells, when followed by transfection significantly enhance ISRE activation to that in and control cells, and when followed by of contrary, poIRF1 knockdown did not alter IFN-β induction after poly(I:C) treatment or SIV, TGEV poly(I:C), ISREofactivation was activation further promoted to promoted a higher to level (Figure On 4A). the On contrary, transfection poly(I:C), ISRE was further a higher level4A). (Figure the and VSV viruses infections, but significantly reduces PRV-induced IFN-β production (Figure 4B). poIRF1 knockdown did not alter IFN-β induction after poly(I:C) treatment or SIV, TGEV and VSV contrary, poIRF1 knockdown did not alter IFN-β induction after poly(I:C) treatment or SIV, TGEV Besides, the ISRE activation after poly(I:C) treatment or virus infections were also significantly and VSV viruses but infections, but significantly reduces PRV-induced IFN-β production 4B). viruses infections, significantly reduces PRV-induced IFN-β production (Figure (Figure 4B). Besides, reduced in poIRF1-knockdown cells (Figure 4C). These data suggest that poIRF1 might be the ISRE after activation aftertreatment poly(I:C) or treatment or virus infections were also significantly theBesides, ISRE activation poly(I:C) virus infections were also significantly reduced in dispensable for IFN-β induction triggered by RNA viruses but be essential for the activation of ISRE. reduced in poIRF1-knockdown cells (Figure 4C). These data suggest that poIRF1 might be poIRF1-knockdown cells (Figure 4C). These data suggest that poIRF1 might be dispensable for IFN-β dispensable for IFN-β induction triggered RNA viruses be essential for the activation of ISRE. induction triggered by RNA viruses but be by essential for thebut activation of ISRE.

Figure 4. Cont. Figure 4. Cont. Figure Cont.

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Figure 4. poIRF1 might dispensablefor for IFN-β IFN-β induction byby RNA viruses but essential Figure 4. poIRF1 might bebe dispensable inductiontriggered triggered RNA viruses but essential for the activation of ISRE. (A) The effect of poIRF1 overexpression on activation of IFN-β and ISRE for the activation of ISRE. (A) The effect of poIRF1 overexpression on activation of IFN-β and ISRE promoter. PK-15/PB and PK-15/IRF1 cells were cotransfected with indicated promoter constructs (0.5 promoter. PK-15/PB and PK-15/IRF1 cells were cotransfected with indicated promoter constructs µg) and pRL-TK (0.025 µg). At 24 h post-transfection, cells were transfected with poly(I:C) for another (0.5 µg) and pRL-TK (0.025 µg). At 24 h post-transfection, cells were transfected with poly(I:C) for 24 h or left untreated and then harvested for detection of IFN-β and ISRE promoter activity. For another 24 h or left untreated and then harvested for detection of IFN-β and ISRE promoter activity. measuring poIRF1-mediated induction of IFN-β, RT-qPCR was used to detect the transcription of For measuring of was IFN-β, RT-qPCRtowas to detect the transcription endogenouspoIRF1-mediated IFN-β.The relativeinduction expression normalized the used expression of GAPDH and of endogenous relative was normalized to cells. the expression of GAPDH expressed asIFN-β.The fold induction relativeexpression to the expression level in control Error bars represent SDs, and expressed fold induction relative toeach the sample expression level in(B,C) control Error barsknockdown represent SDs, whichas were obtained by measuring in triplicate; The cells. effect of poIRF1 which obtained by measuring each sample triplicate; (B,C) The effect of poIRF1 knockdown onwere activation of IFN-β and ISRE promoter. PK-15in cells were transfected with si-IRF1 or si-Ctrl for 36 h. Then, cells were and cotransfected with IFN-β-luc or ISRE-luc and pRL-TK. At 24 h post-transfection, on activation of IFN-β ISRE promoter. PK-15 cells were transfected with si-IRF1 or si-Ctrl for 36 h. withwith poly(I:C) or infected with indicated viruses 12 h or left cells Then,cells cellswere weretransfected cotransfected IFN-β-luc or ISRE-luc and pRL-TK. Atfor 24 hanother post-transfection, untreated and then harvested for detection promoter activity of IFN-β (B) or ISRE (C). were transfected with poly(I:C) or infected with indicated viruses for another 12 h or left untreated and then harvested for detection promoter activity of IFN-β (B) or ISRE (C). 3. Discussion

It is well known that innate immune responses are critical for protecting host cells from early 3. Discussion establishment of virus infection. IRF1 is an important mediator that regulates antiviral innate immune

It is well Although known that innate immune responses areon critical forfunctions protecting host cells from response. many studies have already focused antiviral of human, mouse orearly establishment of virus infection. IRF1 an important mediator that regulates antiviral innate fish IRF1 [20], the role of poIRF1 in is controlling porcine viral infections is poorly understood. In immune this study,Although we showedmany that poIRF1 the on replication of functions four swineof viruses in PK-15 response. studiescould havepotently alreadyinhibit focused antiviral human, mouse or cells and offered an insight into the possible mechanism by which IRF1 exerts its antiviral effects. fish IRF1 [20], the role of poIRF1 in controlling porcine viral infections is poorly understood. In this studies showed the potently broad-spectrum activity of cultureviruses cells under study, weMany showed thathave poIRF1 could inhibitantiviral the replication ofIRF1 fourinswine in PK-15 overexpression or knockdown conditions. According to the literature report, overexpression of IRF1 cells and offered an insight into the possible mechanism by which IRF1 exerts its antiviral effects. can interfere with replication of a wide set of viruses, such as Newcastle disease virus Many studies have showed the broad-spectrum antiviral activity of IRF1 in culture cells under (Paramyxoviridae) [21], vesicular stomatitis virus (Rhabdoviridae) [15,22], encephalomyocarditis virus overexpression or knockdown conditions. According to the literature report, overexpression of (Picornaviridae) [23,24] and hepatitis C virus (Flaviviridae) [12]. Knockdown of IRF1 can enhance the IRF1 replication can interfere with virus replication of [13], a wide setgammaherpesvirus of viruses, such68as Newcastle disease of vaccinia (Poxviridae) murine (Gammaherpesviridae) [14],virus (Paramyxoviridae) [21], vesicular stomatitis[22] virus encephalomyocarditis vesicular stomatitis virus (Rhabdoviridae) and(Rhabdoviridae) West Nile virus[15,22], (Flaviviridae) [23] by using IRF1-virus (Picornaviridae) [23,24] and hepatitis C virus (Flaviviridae) [12]. Knockdown of IRF1 can deficient mice or cells, but have no effect on replication of Newcastle disease virus [21]. In thisenhance study, the we added a new conclusion that poIRF1 could also gammaherpesvirus induce an antiviral state to three other types of [14], replication of vaccinia virus (Poxviridae) [13], murine 68 (Gammaherpesviridae) virus, stomatitis including SIV (Orthomyxoviridae), (Coronaviridae), PRV (Flaviviridae) (Alphaherpesviridae). It isusing vesicular virus (Rhabdoviridae) TGEV [22] and West Nileand virus [23] by worth noting that overexpression of poIRF1 can potently suppress the replication of viruses at In IRF1-deficient mice or cells, but have no effect on replication of Newcastle disease virus [21]. different levels, whereas knockdown of poIRF1 can only promote viral replication moderately or this study, we added a new conclusion that poIRF1 could also induce an antiviral state to three other slightly. Viral replication is strictly controlled by swine IFN-β in PK-15 cells [24–26]. Given the types of virus, including SIV (Orthomyxoviridae), TGEV (Coronaviridae), and PRV (Alphaherpesviridae). regulatory role of IRF1 in the transcription of IFN-β as well as ISGs in vitro, we therefore sought to It is worth noting overexpression of response. poIRF1 can potently suppress the replication of viruses at investigate the that impact of poIRF1 on IFN different As levels, whereas knockdown of poIRF1 canantiviral only promote viral replication moderately one of the ISGs, IRF1 is not a direct-acting effector but a typical modular against or slightly. Viral replication is strictly controlled by swine IFN-β in PK-15 cells [24–26]. Given virus infections. To explore the possible mechanism by which poIRF1 limits the viral replication, we the regulatory role IRF1 in the of IFN-β of as IFN-β well as vitro, weThe therefore detected theofcontribution of transcription poIRF1 to the activation andISGs ISREinpromoter. results sought in to investigate the impact of poIRF1 on IFN response. 6 As one of the ISGs, IRF1 is not a direct-acting antiviral effector but a typical modular against virus infections. To explore the possible mechanism by which poIRF1 limits the viral replication, we detected the contribution of poIRF1 to the activation of IFN-β and ISRE promoter. The results

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in reporter assays showed that overexpression of poIRF1 alone can stimulate the basal activity of the IFN-β promoter, and enhanced the activation of the promoter activity and transcriptional induction of the endogenous IFN-β in response to dsRNA. Our study also revealed that the absence of IRF1 did not clearly affect the induction of IFN-β in PK-15 cells in response to RNA virus infections but impaired the activation of ISRE, indicating that the antiviral phenotype of poIRF1 was not a consequence of IFN-β production and may have arisen directly through ISGs expression. Recently, microarray analysis of IRF1-transduced cells lacking IFN signaling has showed that numerous ISGs were induced by IRF1, supporting the observation that IRF1 can activate IFN-independent pathways to induce antiviral states [17]. Several ISGs have been proposed as the mediators of the antiviral action of IRF1 to restrict replication of diverse virus families, such as Viperin and CH25H [11,14]. However, it is not clear exactly what specific effectors participate in the antiviral process mediated by IRF1. Comparative transcriptome analysis of virus infected wild type and IRF1´{´ cells is planned to identify these antiviral effectors preferentially induced by IRF1 that inhibit viral infections in a virus-type specific manner. Unraveling the antiviral mechanism of these effectors is therefore an important prerequisite to a better understanding of antiviral functions of IRF1. On the other hand, increasing evidence has been presented that constitutive production of IFN-β could be detected in several cells in the absence of viral infection, albeit at a very low level [27,28]. AP-1 and NF-κB components are essential for constitutive IFN-β production [29–32]. Whether poIRF1 is involved in regulation of constitutive expression of IFN-β mediated by AP-1 and NF-κB components remains unclear. Moreover, as the immediate-early type of IFN, IFN-β is induced through the activation of constitutively expressed IRF3 protein in the early phase [33]. Once IFN-β is produced, it binds to its receptor, activates the JAK-STAT signaling pathway, and induces the expression of IRF7 [34]. In the late phase, IRF3 and IRF7 cooperate to amplify the induction of further and wider boost of Type I IFN responses [35]. The different roles of IRF1 in the early and late phases of Type I IFN responses in response to viral infections also require further explorations. In conclusion, our study demonstrated that poIRF1 as an interferon-induced protein plays a significant role in cellular antiviral responses against four kinds of swine viruses, but might be dispensable for IFN-β induction triggered by RNA viruses in PK-15 cells. Given these results, poIRF1 plays potential roles in cellular antiviral responses against swine viruses. 4. Materials and Methods 4.1. Plasmids and Reagents poIRF1(NM_001097413.1) was cloned into pMD-18T vector (TaKaRa, Dalian, China), and then subcloned into PiggyBac (PB) donor vector (System Biosciences, Mountain View, CA, USA). Porcine promoter reporter plasmids, IFN-β-luc and ISRE-luc were constructed according to previous study [36]. Mouse antiserum against poIRF1 and H1N1-HA were prepared previously in our laboratory. Monoclonal antibody against β-actin (Cali-bio, Coachella, CA, USA) was used for Western blot. The double-stranded RNA (dsRNA) analog poly(I:C) (Sigma, St Louis, MO, USA) was used at a final concentration of 2 µg/mL. Cells were transfected with indicated constructs in the presence of Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The target sequences for poIRF1 knockdown and the negative control sequences are shown in Table 1. Small interfering RNAs (siRNA) corresponding to the target sequences were synthesized by a commercial company (Genepharma, Shanghai, China).

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Table 1. Primers or sequences used in this study. Name

poIRF1

GAPDH siRNA

Primer PB-IRF1-F PB-IRF1-R IRF1-Q-F IRF1-Q-R GAPDH-Q-F GAPDH-Q-R si-IRF1 si-Ctrl

Sequence (51 Ñ31 )

Usage

ACTGAATTCATGCCCATCACTCGGATGCGCAT PiggyBac donor TACCTCGAGGGCCTACGGTGCACAAGGAAT vector construction GCACCAGCGACCTGTACAACT RT-qPCR TCCTCATCTGTTGCAGCTTCA TGCCAACGTGTCGGTTGT RT-qPCR TGTCATCATATTTGGCAGGTTT GGGCUGAUCUGGAUUAAUAdTdT Knockdown UUCUCCGAACGUGUCACGUdTdT

4.2. Cells and Viruses PK-15 (Porcine kidney), VERO (African green monkey kidney), and MDCK (Madin-Darby canine kidney) cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and incubated at 37 ˝ C in 5% CO2 . The SIV, VSV, virulent isolate QXX of PRV (PRV-QXX) and TGEV used in this study were isolated from pigs and stored in our laboratory. The attenuated PRV vaccine strain BarthaK61 (PRV-Ba) was obtained from a commercial company (Boehringer-Ingelheim, Ingelheim, Germany). All of these viruses can be grown in PK-15 cells. 4.3. Real-Time Quantitative PCR (RT-qPCR) Cellular RNA extraction was performed using TRIzol by following the standard instructions (Invitrogen, Carlsbad, CA, USA). A volume of 2 µg of RNA was treated with DNase at 37 ˝ C for 30 min to remove potential DNA contamination according to the manufacturer's instructions (Promega, Madison, WI, USA). Reverse transcription was performed using oligo(dT) primers. The specific primers of target genes were designed by Primer 5.0 (Table 1). The quantification of gene transcripts was performed by quantitative PCR using SYBR Green I dye (TOYOBO, Kyoto, Japan). Relative quantitative values of each gene were normalized to the level of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Changes in gene expression were calculated by the ∆∆Ct method. Error bars represent standard deviations (SDs) from three separate experiments with assays performed in triplicate. Differences of gene expression between infected and control cells were analyzed using the Student’s t-test. The results were plotted in graph format as mean ˘ SD. 4.4. Western Blot Cells were washed twice with PBS, lysed with cell lysis buffer (Kangwei, Beijing, China) and incubated on ice for 1 h with vortexing every 15 min and finally centrifuged at 12,000 ˆ g for 30 min at 4 ˝ C. Forty micrograms of protein was loaded per lane into 12% SDS-PAGE gels, separated, and transferred to 0.22 µm nitrocellulose membranes by using semi-dry blotting transfer system. Western blot was performed according to previous study [36]. After washing, bound antibodies were detected with ECL (Thermo, Waltham, MA, USA) by Bio-Imaging System (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). 4.5. Generation of Stable Cell Lines PK-15 cells were co-transfected with donor vector and the helper vector expressing PB transposase. Transfected cells were cultured in fresh medium 36 h post transfection in the presence of puromycin (Merck, Darmstadt, Germany) at a concentration of 5 µg/mL. Puromycin-selective medium was then replaced every 2 days. Puromycin-resistant and GFP-positive cell clones appeared about 10 days after puromycin treatment. A single cell clone was sub-cloned into 96-well plate. As the cell reached confluence, they were split and expanded in 10-cm dishes and saved as cell stocks. The protein expression of poIRF1 in cell clone was determined by RT-qPCR and Western blot.

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4.6. Indirect Immunofluorescence Assay (IFA) PK-15 cells were grown to 70% confluence on coverslips and were treated by poly(I:C) stimulation or SIV infection. At 12 h post-treatment, cells were fixed with 4% paraformaldehyde and permeabilized with ice-cold acetone for 5 min. Cells were treated with a mixture of H1N1-HA antibody and poIRF1 antibody for 1 h at 37 ˝ C in a humidified chamber followed by treatment with secondary, species-specific secondary antibody conjugated with FITC or Cy3 (Molecular Probes) for 1 h. Coverslips containing fixed samples were mounted onto slides using Vectashield containing DAPI (41 ,6-diamidino-2-phenylindole) to allow visualization of cell nuclei (Vector Lab Inc., Burlingame, CA, USA). Confocal images were captured on a LSM510 META microscope (Carl Zeiss, Oberkochen, Germany). 4.7. Antiviral Activity Evaluation For overexpression experiment, PK-15 cells stably expressing poIRF1 or control cells with empty vector alone were plated at a density of 4.0 ˆ 105 cells in 12 well plates. When grown to 80% confluence, cells were incubated with indicated viruses for 1 h and maintained with serum-free DMEM (containing 1 µg/mL TPCK trypsin for SIV) for 24 and 48 h before supernatants were harvested. For knockdown experiment, transfection of siRNA into PK-15 cells was performed by Hirper siRNA transfection reagents according to the manufacturer’s protocol (QIAGEN, Beijing, China). Thirty-six hours after transfection, the cells were incubated with viruses for 1 h and maintained with serum-free DMEM for 24 h before supernatants were harvested. The viral titers in supernatants were measured with 50% tissue culture infectious doses (TCID50 ) or plaque assay (plaque forming unit, PFU) in MDCK or VERO cells. 4.8. Luciferase Reporter Assay To determine the impact of poIRF1 on the activation of IFN-β and ISRE promoter, the luciferase reporter plasmid IFN-β-Luc and ISRE-luc were, respectively, co-transfected in indicated cell lines with pRL-TK vector that provided the constitutive expression of Renilla luciferase (Promega) as an internal control. For knockdown experiment, PK-15 cells were transfected with si-IRF1 or si-Ctrl. After 36 h, the cells were cotransfected with IFN-β-luc or ISRE-luc and pRL-TK. At 24 h post-transfection, cells were transfected with poly(I:C) or infected with indicated viruses for another 12 h or left untreated. Then cells were lysed with 1ˆ passive lysis buffer and the luciferase activity was detected by Dual-Luciferase Reporter Assay System (Promega) and measured with a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA). Transfections were performed in triplicate and repeated at least three times in separate experiments. Error bars represent the mean SDs. Acknowledgments: This work was supported by Program for Innovative Research Team (in Science and Technology) in University of Henan Province (14IRTSTHN015). Special thanks also go to Konrad C. Bradley for critically revising the manuscript. Author Contributions: Yongtao Li, Jun Zhao, Xia Yang and Hongtao Chang conceived and designed the experiments; Yongtao Li, Jun Zhao, Yongxiang Zhao, Lu Chen and Xinwei Wang performed the experiments; Yongtao Li, Chuanqing Wang, Hongying Liu and Xia Yang analyzed the data; and Yongtao Li, Chuanqing Wang and Jun Zhao wrote the paper. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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Antiviral Activity of Porcine Interferon Regulatory Factor 1 against Swine Viruses in Cell Culture.

Interferon regulatory factor 1 (IRF1), as an important transcription factor, is abundantly induced upon virus infections and participates in host anti...
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