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Porcine 2′, 5′-oligoadenylate synthetase 2 inhibits porcine reproductive and respiratory syndrome virus replication in vitro
Accepted Manuscript Porcine 2′, 5′-oligoadenylate synthetase 2 inhibits porcine reproductive and respiratory syndrome virus replication in vitro Mengmeng Zhao, Bo Wan, Huawei Li, Jian He, Xinxin Chen, Linjian Wang, Yinbiao Wang, Sha Xie, Songlin Qiao, Gaiping Zhang PII:
S0882-4010(16)30646-5
DOI:
10.1016/j.micpath.2017.08.011
Reference:
YMPAT 2400
To appear in:
Microbial Pathogenesis
Received Date: 12 October 2016 Revised Date:
8 August 2017
Accepted Date: 9 August 2017
Please cite this article as: Zhao M, Wan B, Li H, He J, Chen X, Wang L, Wang Y, Xie S, Qiao S, Zhang G, Porcine 2′, 5′-oligoadenylate synthetase 2 inhibits porcine reproductive and respiratory syndrome virus replication in vitro, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.08.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Porcine 2′, 5′-oligoadenylate synthetase 2 inhibits porcine reproductive and respiratory syndrome virus replication in vitro Mengmeng Zhao1†, Bo Wan1† Huawei Li2, Jian He1, Xinxin Chen2, Linjian Wang2, Yinbiao
College of Animal Husbandry and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, People’s Republic of China
Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial
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Wang2, Sha Xie2, Songlin Qiao2*, Gaiping Zhang1, 2, 3*
Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, People’s Republic of China 3
Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious
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Diseases and Zoonoses, Yangzhou 225009, People’s Republic of China
*Corresponding author: Gaiping Zhang [email protected]; Songlin Qiao
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[email protected]
Postal address: Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Huayuan Road 116, Jinshui District, 450002, Telephone number:(0371)65738179; Fax number: (0371)65738179
† These authors contributed equally to this work.
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Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is acknowledged a fulminating infectious
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pathogen affecting the pig farming industry, and current vaccines and drugs could hardly inhibit this virus. The 2′, 5′-oligoadenylate synthetase (OASs) have antiviral activities, but the role(s) played by porcine OAS2 in protection against PRRSV infection are unknown. Here we found that endogenous expression of the porcine
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OAS2 gene could be promoted by interferon (IFN)-beta or PRRSV infection in porcine alveolar macrophages.
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Knockdown of porcine OAS2 led to increases in PRRSV replication, and OAS2 expression suppressed replication of PRRSV in a retinoic acid inducible gene I (RIG-I)-dependent manner, anti-PRRSV activity of porcine OAS2 would be lost if RNase L and OAS2 were both silenced. This discovery illustrates a pathway that porcine OAS2 responses to host anti-PRRSV function.
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Keywords: Porcine reproductive and respiratory syndrome virus; 2′, 5′-oligoadenylate synthetase; RIG-1;
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Replication; Porcine alveolar macrophages; RNase L
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ACCEPTED MANUSCRIPT Introduction Porcine reproductive and respiratory syndrome (PRRS) is an important threat to pig farming. Since 2006 a highly pathological PRRS virus (PRRSV), of which main manifestations were high fever, late term abortions,
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reproductive failure, early farrowing, high mortality, respiratory distress, emerged in some Chinese pig farms [1].
PRRSV is the agent of PRRS; it belongs to Arteriviridae family, an enveloped positive-sense RNA virus.
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The length of PRRSV genome is about 15 kb; there are 10 open reading frames (ORFs), between them ORF1a
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and ORF1b encode about 80% of the genome, viral replicase polyproteins are encoded by ORF1a and ORF1b, the viral structural proteins GP2, E, GP3, GP4, GP5, GP5a, M and N were encoded by ORF2a, ORF2b and ORFs 3-7.
PRRSV modulates the host immune responses through suppression of innate immunity and persistent
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infection, previous studies have shown that type I interferon could be inhibited by PRRSV, resulting in PRRSV replication and spread [2, 3]. Limited protection against PRRSV infection is provided by current vaccines and drugs; hence, developing new ways against this pathogen is in an urgent need. Type I interferons (IFNs), such as
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IFN-alpha and IFN-beta, play important roles in antiviral innate immunity and in modulating adaptive immune
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responses to viral infection.
There are many factors effecting the antiviral innate immunity and adaptive immune responses, and interferons are one of the most important one, interferons could blind to the receptors on adjacent cell surfaces, and then JAK/STAT signaling pathway would be activated by interferons, whereby lots of IFN-stimulated genes (ISGs). For example, oligoadenylates synthetase (OAS), ISG15 are induced[4]. Among these ISGs, the 2′, 5′-OAS proteins induced by IFN are present in all vertebrates. The OAS proteins could catalyze the production of 2′, 5′-linked oligoadenylates, the 2′, 5′-linked oligoadenylates are presented
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ACCEPTED MANUSCRIPT with formula pppA-(2, p5’A) n (n>1). RNase L was activated by 2′, 5′-linked oligoadenylates, intracellular 2′, 5′-linked oligoadenylates degraded viral and host RNAs [5]. The 2′, 5′-OAS belongs to a family with low amino acid sequence homology with proteins stimulated by
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interferons. OAS1, OAS2, OAS3, and OASL (OAS-like protein) are four genes encoding four proteins in the OAS family; there is one basal OAS functional unit in OAS1, two basal OAS functional units in OAS2, three basal OAS functional units in OAS3, a tandem ubiquitin unit in the C-terminal domain of OASL enzyme that
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lacks 2′, 5′-OAS activity [6-10]. OAS1 and OASL are distributed widely in vertebrates, whereas OAS2 and
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OAS3 proteins could only be found in mammals [11].
The porcine OAS family contains OAS1a, OAS1b, OAS2, and OASL, but no OAS3 [12]. The amino acid sequence homology between OAS1a and OAS1b is 89.1% [8], and porcine OAS1a was the first to be crystallized [8]. OAS1a shares 73% nucleotide sequence identity with human OAS1 , enzymatic characteristics
mouse OASs [13].
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could be found in OAS1a, while high sequence homology was found between porcine OASs and human and
Antiviral effects of OAS proteins have been demonstrated in many researches. OAS1b, for example, was
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able to restrict West Nile virus (WNV) replication [14, 15], but this effect was not modulated by the OAS/RNase
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L pathway [16, 17]. Additionally, while human OAS1 and OAS3 can inhibit dengue virus [18], OAS1 and OAS3 can inhibit hepatitis C virus [18, 19], but the effect would be lost without effect of RNase L. The single nucleotide polymorphism could be responsible for increasing the probability of WNV disease [18], and while extracellular OAS1 possesses strong antiviral activity [20]. Although the antiviral effects of other OAS proteins have been well-studied, it remains unclear whether porcine OAS2 protein can inhibit PRRSV. In this paper we report the discovery of antiviral characteristics of porcine OAS2 against PRRSV; the results indicate that porcine OAS2 has antiviral activity against PRRSV.
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ACCEPTED MANUSCRIPT Material and methods 2.1. Cell lines and virus Porcine alveolar macrophages (PAMs) were obtained from the lungs of seven-week-old PRRSV-free pigs;
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they were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS). CRL-2843-CD163 cells were obtained from Professor Enmin Zhou of Northwest A & F University, China[21]. The CRL-2843-CD163 cells were maintained in RPMI 1640 medium supplemented with 6% FBS
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(GIBCO, Invitrogen, USA) at 37℃ in 5% CO2. The CRL-2843-CD163 cells were modified from CRL-2843
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cells, via CD163 gene transfection into CRL-2843 cells, to confer PRRSV with the ability to infect the CRL-2843-CD163 cells.
The PRRSV BJ-4 strain was provided by Professor Hanchun Yang from China Agricultural University. We strictly followed the rules for animal care which have been approved by the State Council of the People’s
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Republic of China [22-24].
2.2 Analysis of OAS2 in PAMs which are infected with BJ-4 PRRSV PAMs were infected with PRRSV BJ-4 at a multiplicity of infection (MOI) of 1 or 0.1 for the times
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indicated (i.e., 0, 6, 24, 36, 48 h), after which PAMs were collected and subjected to quantitative real-time PCR
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(qRT-PCR) to analyze the OAS2 expression levels.
2.3 Interferon treatment
The PAMs were treated with 1000 IU/ml of IFN-beta (Peprotech, Rocky Hill, NJ, USA) for the times indicated (i.e., 0, 6, 12, 24, 36, and 48), after which the cells were collected and subjected to qRT-PCR to analyze the OAS2 expression levels.
2.4 Plasmid construction The porcine OAS2 gene was amplified from cDNA from the PAMs using the following primer sequences:
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2.5 RNA interference Nonsense siRNA, OAS2 siRNA, retinoic acid inducible gene I (RIG-1) siRNA and RNase L siRNA were designed and synthesized by GenePharma Co., Ltd (Suzhou, China).
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2.6 Cell transfection and viral infections
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CRL-2843-CD163 cells were maintained in 24-well plates and, when the cell monolayer reached 60-70% confluence, 800 ng of the pFLAG-pOAS2 expression vector or the control expression vector (p3xFLAG-CMV -7.1) were transfected into the cells using lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. After 24 h, the cells were infected with PRRSV BJ-4 (MOI=1) for another 24 h, and
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the mRNA levels of PRRSV N (ORF7), OAS2, interferon regulatory factor (IRF)3, IRF7, Toll-like receptor (TLR)3, TLR7, RIG-1, melanoma differentiation-associated protein 5 (MDA5), myeloid differentiation primary response 88 (MyD88), TANK binding kinase 1 (TBK1), nuclear factor-κB (NF-κB), interleukin (IL)-8, IL-1
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western blotting.
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beta, IFN-alpha, and IFN-beta mRNAs were detected by qRT-PCR. IRF3 and pIRF3 proteins were detected by
PAMs were transfected with 60 nM of OAS2 siRNA, RNase L siRNA, RIG-1 siRNA or nonsense siRNA, and then infected with PRRSV BJ-4 (MOI=1) for another 24 h, and PRRSV N mRNA was detected by qRT-PCR.
CRL-2843-CD163 cells were transfected with 60 nM of RNase L siRNA, RIG-1 siRNA or nonsense siRNA and, the CRL-2843-CD163 cells, which RNase L or RIG-1 were knockdown, were transfected with pFLAG-pOAS2 or the negative control vector, and infected with BJ-4 (MOI=1) for another 24 h, after which
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ACCEPTED MANUSCRIPT PRRSV N mRNA was detected by qRT-PCR.
2.7 Cell viability assays The enhanced Cell Counting Kit-8 assay (Beyotime Biotech, China) here was used to evaluate cytotoxicity
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levels, whereby pFLAG-pOAS2 or p3xFLAG-CMV TM-7.1 expression vectors (800 ng each) were transfected individually into CRL-2843-CD163 cells plated in 24-well plates, and the cytotoxicity of OAS2 was evaluated. PAMs plated in 24-well plates were transfected with 60 nM of OAS2 siRNA, RNase L siRNA, RIG-1
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siRNA or nonsense siRNA for 48 h, the cytotoxicity was evaluated as stated above.
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2.8 Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA from the cells was treated with TRIzol (Invitrogen, USA). Reverse transcriptase reactions were performed using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Dalian, China), while SYBR Ex Taq™ (Takara, Dalian, China) was used in the polymerase chain reactions, and the machine was the 7500 Fast
2.9 Western blotting
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Real-time PCR System (Applied Biosystems, Foster city, CA).
CRL-2843-CD163 cells were seeded in 24-well plates and then transfected with the pFLAG-OAS2
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expression vector, and then infected with PRRSV BJ-4 (MOI=1). 48 h later, western blotting was performed as
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described previously [25] using either an anti-Flag monoclonal antibody (1:200, Abnova, Taipei, Taiwan) , an anti-GAPDH monoclonal antibody (1:1000, Beijing Biosynthesis Biotech Co., Ltd, China), an anti-IRF3 monoclonal antibody (1:1000, Proteintech Group, China), or an anti-pIRF3 monoclonal antibody (1:1000, Cell Signaling Technology, USA). The membranes were washed three times with Tris-buffered saline containing Tween-20 (TBST), and then reacted with
horseradish peroxide-conjugated goat anti-rabbit IgG (1:10000,
Zhongshan Golden bridge Biotechnology, China) or horseradish peroxide-conjugated goat anti-mouse IgG (1:10000, Jackson ImmunoResearch, West Grove, PA, USA) for one hour at 37℃, and then washed with TBST
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2.10 Viral titers
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Supernatants from the transfected groups were obtained at 48 h post infection, and PRRSV titers were measured.
2.11 Luciferase reporter assays
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CRL-2843-CD163 cells were seed in 24 well-plates, when they reached 70% confluence, 200 ng of
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pIFN-Beta-Luc (luciferase reporter plasmid), 80 ng of pRL-TK, and the expression vector (500 ng) or the control vector were transfected into the cells with lipofectamine 2000 (Invitrogen, USA). 24 h later, these cells were transfected with 1.5 µg Poly (I:C) for 9 h, Luciferase Reporter System (Promega, Madison, WI, USA) was used to detect the luciferase activity, and the CRL-2843-CD163 cells were also
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infected with PRRSV for 24 hours(MOI=1), and luciferase reporter assays were performed.
2.12 Secreted IFN-beta protein measurements
procedures.
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The secreted IFN-beta was detected with an ELISA kit (Cloud-Clone Group, China) by the manufacturer’s
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2.13 Statistical analysis
Every experiment has three independent experiments; each independent experiment has three technical replicates. GraphPad Prism 5 was used to analyze the results using Student's t-test.
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ACCEPTED MANUSCRIPT Results 3.1 IFN-beta and PRRSV induce porcine OAS2 mRNA expression in PAMs The results showed that from 6 hours post treatment with 1000 IU/ml IFN-beta, porcine OAS2 mRNA
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expression level increased by 40-fold in PAMs (Fig. 1A). The peak mRNA expression level for the OAS2 gene occurred at 12 h post treatment in the PAMs.
The expression kinetics of OAS2 in the PAMs stimulated by PRRSV was also investigated. 24 h later, the
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following PRRSV treatment resulted in only an 8-fold increase.
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highest level of OAS expression following PRRSV infection was detected (Fig. 1B). Induction of OAS2 mRNA
3.2 OAS2 overexpression inhibits PRRSV replication in CRL-2843-CD163 cells Immunoblotting revealed that porcine OAS2 was successfully expressed in the CRL-2843-CD163 cells transfected with the pFLAG-pOAS2 expression vector (Fig. 1C). The transfection efficiency of the plasmid (800
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ng) in the 24-well-plate cells was about 25 %, and no significant cytotoxic effect was observed. Relative expression of PRRSV ORF7 mRNA of CRL-2843-CD163 cells transfected with pFLAG-pOAS2 was lower (Fig. 1D). We also found that the TCID50 of the supernatants from cells transfected with
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pFLAG-pOAS2 was lower (Fig. 1E).
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As shown in Fig. 2A, after infection with PRRSV, OAS2 induced a 2.2-fold increase in IFN-alpha mRNA, a 2.0-fold increase in IFN-beta mRNA, a 2.4-fold increase in IRF3 mRNA, a 4.3-fold increase in TLR-3 mRNA, a 4.3-fold increase in RIG-1 mRNA, a 2.3-fold increase in NF-κB mRNA, a 2.6-fold increase in MDA5 mRNA, a 6.3-fold increase in MyD88 mRNA, a 1.9-fold increase in TBK1 mRNA, and no increases in TNF-alpha, IL-8, IL-1beta, IRF7 mRNA levels. In response to PRRSV infection, the supernatant from the CRL-2843-CD163 cells transfected with pFLAG-pOAS2 had a higher concentration of IFN-beta than that of the CRL-2843-CD163 cells transfected with
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ACCEPTED MANUSCRIPT the p3xFLAG-CMV -7.1 vector (Fig. 2C). The active and latent forms of IRF-3 were also analyzed by western blotting, and as shown in Fig. 2D, OAS2 enhanced the phosphorylation level of IRF-3. IFN-beta promoter activity got significantly enhanced by OAS2 induced by Poly (I:C) and PRRSV (Fig. 2E).
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3.3 Knockdown of OAS2 led to increases in PRRSV replication in PAMs Efficiency of OAS2 gene knockdown in PAMs was approximately 70% (Fig. 3A), while PRRSV ORF7 mRNA increased significantly in the OAS2 knockdown group (Fig. 3D). The TCID50 of the supernatants from
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the OAS2 knockdown group was higher (Fig. 3C).
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In siRNA rescue experiments, 60 nM of si-OAS2, in combination with 800 ng of the pFLAG-pOAS2 plasmid were transfected into the CRL-2843-CD163 cells, and 24 h later, cells were infected with PRRSV. The results showed that no increase in the PRRSV ORF7 mRNA expression level was found.
3.4 RNase L- and RIG-1-dependent antiviral activities
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In the PAMs, 60 nM of RNase L siRNA induced a 33% knockdown of RNase L mRNA (Fig. 4A), which in turn induced a 1.9-fold increase in PRRSV ORF7 mRNA (Fig. 4B). In order to detect whether the anti-PRRSV function rely on RNase L, 30 nM of OAS2 siRNA and 30 nM of RNase L siRNA were co-transfected into PAMs,
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followed by infection with PRRSV (MOI=1). In comparison with the control, no increase in PRRSV ORF7
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mRNA expression in the OAS2/RNase L co-silenced group was found (Fig. 4C). For the PAMs, 60 nM of RIG-1 siRNA induced a 30% knockdown of RIG-1 mRNA (Fig.4D), and this facilitated a 1.7-fold increase in PRRSV ORF7 mRNA (Fig. 4E). To determine whether the anti-PRRSV activity was dependent upon the RIG-1 pathway, both of the OAS2 siRNA and RIG-1 siRNA were transfected into PAMs, and then PRRSV infect the cells (MOI=1). We found no increase in PRRSV ORF7 mRNA expression in the OAS2/ RIG-1 co-silencing group (Fig. 4F). In the CRL-2843-CD163 cells, 60 nM of RNase L siRNA induced a 29% knockdown of RNase L mRNA
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ORF7 mRNA expression in the RNase L knockdown group was observed (Fig. 4I). In the CRL-2843-CD163 cells, 60 nM of RIG-1 siRNA induced a 28% knockdown of RIG-1 mRNA (Fig.4L), and this facilitated a 1.6-fold increase in PRRSV ORF7 mRNA (Fig. 4M). To determine whether the
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anti-PRRSV activity was dependent on the RIG-1 pathway, CRL-2843-CD163 cells with RIG-1 knocked-down
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were transfected with pFLAG-pOAS2 or the negative control vector. No increase in PRRSV ORF7 mRNA
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expression in the RNase L knockdown group was observed (Fig. 4N).
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ACCEPTED MANUSCRIPT Discussion The current vaccines could hardly inhibit PRRSV infection, there are some factors: high frequency of PRRSV genome mutation, antibody dependent enhancement (ADE) effect, and persistence of virus in the
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infected pigs. So finding new antiviral therapy becomes a useful method to control PRRSV. Up to now, some ISGs are reported to inhibit viruses, for example, IFIT3[26, 27], ISG15[4], Mx2[28], but the effect of OAS were not well-known.
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This paper has shown PRRSV replication was significantly inhibited by overexpression of porcine OAS2,
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but increased following knockdown of porcine OAS2. The antiviral mechanism of OAS is shown in some other researches, but not well understood, overexpression of porcine OAS2 could trigger RIG-I and some other molecules, it has been shown human OASL binds RIG-I and has antiviral activities [29]. Porcine OAS2, which has also been found to have antiviral activities, can also activate the RIG-I pathway, and the innate immune
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character could share sort of similarity for pigs and humans in this respect. During the experiments, we found that some antibodies used for human signaling molecules could not be used in the porcine signaling molecules, maybe some amino acids mutations change the recognition clues for porcine signaling molecules, so this
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become a question for the porcine innate immunes.
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Zheng et al reported that porcine OAS2 can inhibit JEV, via porcine OAS2 functioning through OAS/RNase L signal channel [30], our result consistent with that found for JEV. For the porcine OAS1, we found that overexpressions of OAS1 also inhibit PRRSV, but the effect is not as significant as that of porcine OAS2. Knockdown of OAS1 could increase the replication of PRRSV, and also the effect was not as good as that of porcine OAS2 (date not shown), so we focused on antiviral effect of porcine OAS2 only. We first discovered that porcine OAS2 could induce the phosphorylation of IRF3 to enhance IFN pathway. The host gene could inhibit the virus through this way, there are reports showing that mouse OASL1 could
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ACCEPTED MANUSCRIPT inhibit type I interferon [31], this is different from the porcine OAS, and the resultant innate immune character did not share similarity between pigs and mouse in this respect. Porcine OAS2 has enzymatic and antiviral activities, whether its antiviral activities connect with enzymatic activities needs further research. The porcine
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OAS2 located in the cytoplasm (date not shown), the PRRSV also replicate in the cytoplasm, whether the porcine OAS2 interacted with viral proteins in the cytoplasm needs more research, maybe the PRRSV could
are required for the antivirus activity needs more research.
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escape the innate immunity through inhibiting OAS2 or other OAS, and which critical regions or amino acids
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PRRSV can replicate in the Marc-145 cells which are derived from monkey kidney[32], and in PAMs which are target porcine cells in vivo [33], but PAMs are not easy for plasmid DNA to be transfected, so we used CRL-2843-CD163 cells to investigate the antiviral activity of OAS2 and found that the transfection efficiency of 800 ng of OAS2 in 24-well-plates cells was about 30%. The CRL-2843-CD163 cell line was
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modified from CRL-2843 cells, which involved CD163 transfection into the CRL-2843 cells. Thus, PRRSV has the ability to infect CRL-2843-CD163 cells, a result consistent with that obtained for CRL-2843-CD163 cells and PAMs, which highlights the utility of CRL-2843-CD163 cells for studying the pathogenesis of PRRSV;
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therefore, use of the CRL-2843-CD163 line could sidestep the need for proper preparation of PAMs.
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Given the antiviral activities of OAS2 against RNA viruses [19, 34, 35], it is possible that OAS2 is required for the prevention of RNA viruses [36]. Therefore, it seems that OAS2 is a universal pattern recognition receptor for the defense against invasion with DNA and RNA viruses. In summary, after cloning of porcine OAS2 gene, it was expressed in CRL-2843-CD163 cells. Porcine OAS2 is an inhibitor to PRRSV; it could increase IFN-beta expression. Further studies on the structure and function of porcine OAS2 mediated signaling pathway are needed, maybe these research could help find new ways to control PRRSV infection.
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Acknowledgment: Our work received funds from the National Natural Science Fund (No. 31490601,
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31502040), and the National Program on the Key Basic Research Project (2014CB542702).
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[24] Wang LJ, Xie W, Chen XX, Qiao S, Zhao M, Gu Y, et al. Molecular epidemiology of porcine reproductive and respiratory syndrome virus in Central China since 2014: The prevalence of NADC30-like PRRSVs. Microbial pathogenesis. 2017;109:20-8. [25] Zhao M, Qian J, Xie J, Cui T, Feng S, Wang G, et al. Characterization of polyclonal antibodies against nonstructural protein 9 from the porcine reproductive and respiratory syndrome virus. Frontiers of Agricultural Science and Engineering. 2016;3:153-60. [26] Miller LC, Jiang Z, Sang Y, Harhay GP, Lager KM. Evolutionary characterization of pig interferon-inducible transmembrane gene family and member expression dynamics in tracheobronchial lymph nodes of pigs infected with swine respiratory disease viruses. Veterinary immunology and immunopathology. 2014;159:180-91. [27] Zhang L, Liu J, Bai J, Du Y, Wang X, Liu X, et al. Poly(I:C) inhibits porcine reproductive and respiratory syndrome virus replication in MARC-145 cells via activation of IFIT3. Antiviral research. 2013;99:197-206. [28] Wang H, Bai J, Fan B, Li Y, Zhang Q, Jiang P. The Interferon-Induced Mx2 Inhibits Porcine Reproductive
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ACCEPTED MANUSCRIPT and Respiratory Syndrome Virus Replication. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 2016;36:129-39. [29] Zhu J, Zhang Y, Ghosh A, Cuevas RA, Forero A, Dhar J, et al. Antiviral activity of human OASL protein is mediated by enhancing signaling of the RIG-I RNA sensor. Immunity. 2014;40:936-48. [30] Zheng S, Zhu D, Lian X, Liu W, Cao R, Chen P. Porcine 2', 5'-oligoadenylate synthetases inhibit Japanese encephalitis virus replication in vitro. Journal of medical virology. 2016;88:760-8. [31] Lee MS, Kim B, Oh GT, Kim YJ. OASL1 inhibits translation of the type I interferon-regulating
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Figure Legends
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Fig. 1 OAS2 overexpression inhibits PRRSV replication (A) PAMs were stimulated by IFN-beta, at different time points (6, 12, 24, 36, and 48); PAMs were subjected to
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qRT-PCR to quantify OAS2 mRNA levels. (B) PRRSV infected the PAMs, at different time points (6, 12, 24, 36, and 48); PAMs were subjected to qRT-PCR to quantify OAS2 mRNA levels. (C) CRL-2843-CD163 cells were transfected with the pFLAG-pOAS2 vector, 48 h later; western blotting was performed to detect expression. (D) pFLAG-pOAS2 or the negative control vectors were transfected into CRL-2843-CD163 cells; 24 h later, PRRSV infected the cells. 48 h later, qRT-PCR was used to measure the PRRSV ORF7 mRNA expression (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (E) TCID50 was used to measure the viral titers (* means a P value was less than 0.05, 17
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Fig. 2 OAS2 enhances RIG-1 signaling
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significant differences. ** means a P value was less than 0.01, very significant differences).
(A) pFLAG-pOAS2 were transfected into CRL-2843-CD163 cells; 24 h later PRRSV infected the cells, 48 h
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later qRT-PCR was used to measure IFN-alpha, IFN-beta, IL-8, IL-1beta, and TNF-alpha mRNA expression levels (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (B) qRT-PCR was performed to measure IFR3, IFR7, TLR3, TLR7, RIG-1, NF-κB,
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MDA5, MyD88, and TBK1 mRNA expression levels (* means a P value was less than 0.05, significant
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differences. ** means a P value was less than 0.01, very significant differences). (C) ELISA was performed to measure IFN-beta protein levels (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (D) Immunoblotting was used to analyze the cells with anti-IRF-3 and anti-pIRF-3 antibodies. (E) OAS2 overexpression enhanced the activity of the IFN-beta promoter. On day 0, the CRL-2843-CD163 cells were cultured. On day 1, 200 ng of pIFN-Beta-Luc, 80 ng of pRL-TK, and the expression vector (500 ng) or the control were transfected into the cells. On day 2, 1.5 µg of Poly (I:C) treated the cells, 9 h later IFN-beta promoter activities were assessed (* means a P value was less than 0.05,
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ACCEPTED MANUSCRIPT significant differences. ** means a P value was less than 0.01, very significant differences). PRRSV also infected cells (MOI=1), and on day 3, luciferase reporter assays were performed, followed by cell harvesting 24 h later (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01,
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very significant differences).
Fig. 3 OAS2 knockdown in PAMs enhances PRRSV replication (A) 60 nM of OAS2 siRNA (si-OAS2) or negative control siRNA (si-NC) were transfected into PAMs, 24 h later, PRRSV infected the cells for 48h (MOI=1). qRT-PCR was used to measure OAS2 expression (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (B) Evaluation of cell cytotoxicity of si-OAS2 in PAMs. (C) Viral titers were investigated via 19
ACCEPTED MANUSCRIPT TCID50 (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (D) qRT-PCR was used to measure PRRSV N expression levels (* means a P value
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was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences).
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Fig. 4 The anti-PRRSV activity of OAS2 is dependent on RNase L and RIG-1
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ACCEPTED MANUSCRIPT (A) PAMs were transfected with 60nM of RNase L siRNA (si- RNase L) or negative control siRNA (si-NC); At 24 h past transfection, PRRSV infected the cells, RNase L expression levels were measured by qRT-PCR (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very
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significant differences). (B) RRSV N mRNA expression levels were measured by qRT-PCR (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (C) 30nM of RNase L siRNA and 30nM of OAS2 siRNA were co-transfected into PAMs; 24 h later, cells were
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infected with PRRSV (MOI=1), RRSV N mRNA expression levels were measured by qRT-PCR. (D) PAMs
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were transfected with 60nM of RNase L siRNA (si- RIG-1) or negative control siRNA (si-Ctrl); 24 h later, cells were infected with PRRSV (MOI=1), RNase L mRNA expression levels were measured by qRT-PCR (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (E) qRT-PCR was used to measure RRSV N mRNA expression levels (* means a P
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value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (F) 30nM of RIG-1 siRNA and 30nM of OAS2 siRNA were co-transfected into PAMs; 24 h later, cells were infected with PRRSV (MOI=1), RRSV N mRNA expression levels were measured by qRT-PCR.
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(G) CRL-2843-CD163 cells were transfected with 60nM of RNase L siRNA (si- RNase L) or negative control
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siRNA (si-NC); 24 h later, PRRSV was inoculated at a MOI of 1 for 48 h. qRT-PCR was used to measure RNase L expression levels (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (H) qRT-PCR was used to measure RRSV N mRNA expression levels (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (I) CRL-2843-CD163 cells were transfected with 60nM of RNase L siRNA; 24 h later, the CRL-2843-CD163 cells with RNase L knocked down were transfected with 400 ng of pFLAG-pOAS2 or the negative control vector, and cells were infected with PRRSV, qRT-PCR was used to measure RRSV N mRNA
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ACCEPTED MANUSCRIPT expression levels. (L) CRL-2843-CD163 cells were transfected with 60nM of RNase L siRNA (si- RNase L) or negative control siRNA (si-NC); 24 h later, cells were infected with PRRSV. qRT-PCR was used to measure RNase L expression (* means a P value was less than 0.05, significant differences. ** means a P value was less
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than 0.01, very significant differences). (M) qRT-PCR was used to measure RRSV N mRNA expression levels (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (N) CRL-2843-CD163 cells were transfected with 60nM of RIG-1 siRNA,24 h later, the
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CRL-2843-CD163 cells with RIG-1 knocked down were transfected with 400 ng of pFLAG-pOAS2 or the
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negative control vector, and cells were infected with PRRSV, qRT-PCR was used to measure RRSV N mRNA
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expression levels.
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ACCEPTED MANUSCRIPT Table1 Primers used in the research primer sequence TATGAATTCAATGGGAAACTGGGGGTCCC
OAS2- R
CGCGACGACTCAGTCCATGAATCTCC
qOAS2-F
ATGGTGTGAACGCAAACTGA
qOAS2-R
AACCTTCAGCCGTGTCAAAG
qGAPDH-F
CTGCCGCCTGGAGAAACCT
qGAPDH-R
GCTGTAGCCAAATTCATTGTCG
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OAS2- F
qIRF3-F
CCACCAAACUGAAGGAUUU AAAUCCUUCAGUUUGGUGG GCCCUUAACCAAGCAGGUUTT AACCUGCUUGGUUAAGGGCTT UGGAAGAGAUGAAUGCAUA UAUGCAUUCAUCUCUUCCA AAGGTTGTCCCCATGTGTCTCCG
qIRF3-R
GGAAATGTGCAGGTCCACCGTG
qIRF7-F
TCCAGCCGAGATGCTAAGTG
qIRF7-R
GTCCAAGTCCTGCCCGATGT
qORF7-F
AAACCAGTCCAGAGGCAAGG
qORF7-R
GCAAACTAAACTCCACAGTGTAA
qIFN-beta -F
CTAGCACTGGCTGGAATGAGACT
Si-OAS2
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Si-RIG-1
qIFN-beta -R
GGCCTTCAGGTAATGCAGAATC
qTNF-alpha-F
qIL-8-R
ACGGGCTTATCTGAGGTTTGAG
GGCAGTTTTCCTGCTTTCT CAGTGGGGTCCACTCTCAAT
CAGAGCAGCGGCGGAATC
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qRIG-1-F
CACCACGCTCTTCTGCCTAC
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qTNF-alpha-R qIL-8-F
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Si-RNase L
qRIG-1-R
ACTCAAGGTTGCCCAT
qTLR7-F
GAACTGTTTC TCTACAACA
qTLR7-R
AGACTTGTAATTCTGTCA
qTLR3-F
TACTGTACAC AACTTCTACC
qTLR3-R
TTAAATCCTCCATCCAAGG
qNF-κB-F
CCAGCACCTCCACTCCATTC
qNF-κB-R
ACATCAGCACCCAAAGACACC
qMDA5-F
CGAATTAACAGGCACCGATT
qMDA5-R
CGTCCAGACTTGGCTGATCT
qMyD88-F
CTCCGGAGCG GAGTCCGCG
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qTBK1-F
CCAGTGGAT GTTCAAAT
qTBK1-R
CTCCCACATGGACAAAAT
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qMyD88-R
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Highlights We firstly found that porcine 2’, 5’-oligoadenylate synthetase (OAS2) could inhibit the PRRSV replication, and the inhibition was dependent by RNase L. Porcine OAS2 suppressed replication of PRRSV in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. Co-silencing of RNase L and OAS2 revealed that the anti-PRRSV function of porcine OAS2 was RNase L dependent. Our findings show a mechanism by which porcine OAS2 contributes to host anti-PRRSV responses by enhancing RIG-I activation.
S0882-4010(16)30646-5
DOI:
10.1016/j.micpath.2017.08.011
Reference:
YMPAT 2400
To appear in:
Microbial Pathogenesis
Received Date: 12 October 2016 Revised Date:
8 August 2017
Accepted Date: 9 August 2017
Please cite this article as: Zhao M, Wan B, Li H, He J, Chen X, Wang L, Wang Y, Xie S, Qiao S, Zhang G, Porcine 2′, 5′-oligoadenylate synthetase 2 inhibits porcine reproductive and respiratory syndrome virus replication in vitro, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.08.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Porcine 2′, 5′-oligoadenylate synthetase 2 inhibits porcine reproductive and respiratory syndrome virus replication in vitro Mengmeng Zhao1†, Bo Wan1† Huawei Li2, Jian He1, Xinxin Chen2, Linjian Wang2, Yinbiao
College of Animal Husbandry and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, People’s Republic of China
Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial
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Wang2, Sha Xie2, Songlin Qiao2*, Gaiping Zhang1, 2, 3*
Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, People’s Republic of China 3
Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious
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Diseases and Zoonoses, Yangzhou 225009, People’s Republic of China
*Corresponding author: Gaiping Zhang [email protected]; Songlin Qiao
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[email protected]
Postal address: Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Huayuan Road 116, Jinshui District, 450002, Telephone number:(0371)65738179; Fax number: (0371)65738179
† These authors contributed equally to this work.
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Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is acknowledged a fulminating infectious
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pathogen affecting the pig farming industry, and current vaccines and drugs could hardly inhibit this virus. The 2′, 5′-oligoadenylate synthetase (OASs) have antiviral activities, but the role(s) played by porcine OAS2 in protection against PRRSV infection are unknown. Here we found that endogenous expression of the porcine
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OAS2 gene could be promoted by interferon (IFN)-beta or PRRSV infection in porcine alveolar macrophages.
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Knockdown of porcine OAS2 led to increases in PRRSV replication, and OAS2 expression suppressed replication of PRRSV in a retinoic acid inducible gene I (RIG-I)-dependent manner, anti-PRRSV activity of porcine OAS2 would be lost if RNase L and OAS2 were both silenced. This discovery illustrates a pathway that porcine OAS2 responses to host anti-PRRSV function.
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Keywords: Porcine reproductive and respiratory syndrome virus; 2′, 5′-oligoadenylate synthetase; RIG-1;
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Replication; Porcine alveolar macrophages; RNase L
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ACCEPTED MANUSCRIPT Introduction Porcine reproductive and respiratory syndrome (PRRS) is an important threat to pig farming. Since 2006 a highly pathological PRRS virus (PRRSV), of which main manifestations were high fever, late term abortions,
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reproductive failure, early farrowing, high mortality, respiratory distress, emerged in some Chinese pig farms [1].
PRRSV is the agent of PRRS; it belongs to Arteriviridae family, an enveloped positive-sense RNA virus.
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The length of PRRSV genome is about 15 kb; there are 10 open reading frames (ORFs), between them ORF1a
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and ORF1b encode about 80% of the genome, viral replicase polyproteins are encoded by ORF1a and ORF1b, the viral structural proteins GP2, E, GP3, GP4, GP5, GP5a, M and N were encoded by ORF2a, ORF2b and ORFs 3-7.
PRRSV modulates the host immune responses through suppression of innate immunity and persistent
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infection, previous studies have shown that type I interferon could be inhibited by PRRSV, resulting in PRRSV replication and spread [2, 3]. Limited protection against PRRSV infection is provided by current vaccines and drugs; hence, developing new ways against this pathogen is in an urgent need. Type I interferons (IFNs), such as
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IFN-alpha and IFN-beta, play important roles in antiviral innate immunity and in modulating adaptive immune
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responses to viral infection.
There are many factors effecting the antiviral innate immunity and adaptive immune responses, and interferons are one of the most important one, interferons could blind to the receptors on adjacent cell surfaces, and then JAK/STAT signaling pathway would be activated by interferons, whereby lots of IFN-stimulated genes (ISGs). For example, oligoadenylates synthetase (OAS), ISG15 are induced[4]. Among these ISGs, the 2′, 5′-OAS proteins induced by IFN are present in all vertebrates. The OAS proteins could catalyze the production of 2′, 5′-linked oligoadenylates, the 2′, 5′-linked oligoadenylates are presented
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ACCEPTED MANUSCRIPT with formula pppA-(2, p5’A) n (n>1). RNase L was activated by 2′, 5′-linked oligoadenylates, intracellular 2′, 5′-linked oligoadenylates degraded viral and host RNAs [5]. The 2′, 5′-OAS belongs to a family with low amino acid sequence homology with proteins stimulated by
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interferons. OAS1, OAS2, OAS3, and OASL (OAS-like protein) are four genes encoding four proteins in the OAS family; there is one basal OAS functional unit in OAS1, two basal OAS functional units in OAS2, three basal OAS functional units in OAS3, a tandem ubiquitin unit in the C-terminal domain of OASL enzyme that
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lacks 2′, 5′-OAS activity [6-10]. OAS1 and OASL are distributed widely in vertebrates, whereas OAS2 and
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OAS3 proteins could only be found in mammals [11].
The porcine OAS family contains OAS1a, OAS1b, OAS2, and OASL, but no OAS3 [12]. The amino acid sequence homology between OAS1a and OAS1b is 89.1% [8], and porcine OAS1a was the first to be crystallized [8]. OAS1a shares 73% nucleotide sequence identity with human OAS1 , enzymatic characteristics
mouse OASs [13].
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could be found in OAS1a, while high sequence homology was found between porcine OASs and human and
Antiviral effects of OAS proteins have been demonstrated in many researches. OAS1b, for example, was
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able to restrict West Nile virus (WNV) replication [14, 15], but this effect was not modulated by the OAS/RNase
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L pathway [16, 17]. Additionally, while human OAS1 and OAS3 can inhibit dengue virus [18], OAS1 and OAS3 can inhibit hepatitis C virus [18, 19], but the effect would be lost without effect of RNase L. The single nucleotide polymorphism could be responsible for increasing the probability of WNV disease [18], and while extracellular OAS1 possesses strong antiviral activity [20]. Although the antiviral effects of other OAS proteins have been well-studied, it remains unclear whether porcine OAS2 protein can inhibit PRRSV. In this paper we report the discovery of antiviral characteristics of porcine OAS2 against PRRSV; the results indicate that porcine OAS2 has antiviral activity against PRRSV.
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ACCEPTED MANUSCRIPT Material and methods 2.1. Cell lines and virus Porcine alveolar macrophages (PAMs) were obtained from the lungs of seven-week-old PRRSV-free pigs;
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they were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS). CRL-2843-CD163 cells were obtained from Professor Enmin Zhou of Northwest A & F University, China[21]. The CRL-2843-CD163 cells were maintained in RPMI 1640 medium supplemented with 6% FBS
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(GIBCO, Invitrogen, USA) at 37℃ in 5% CO2. The CRL-2843-CD163 cells were modified from CRL-2843
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cells, via CD163 gene transfection into CRL-2843 cells, to confer PRRSV with the ability to infect the CRL-2843-CD163 cells.
The PRRSV BJ-4 strain was provided by Professor Hanchun Yang from China Agricultural University. We strictly followed the rules for animal care which have been approved by the State Council of the People’s
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Republic of China [22-24].
2.2 Analysis of OAS2 in PAMs which are infected with BJ-4 PRRSV PAMs were infected with PRRSV BJ-4 at a multiplicity of infection (MOI) of 1 or 0.1 for the times
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indicated (i.e., 0, 6, 24, 36, 48 h), after which PAMs were collected and subjected to quantitative real-time PCR
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(qRT-PCR) to analyze the OAS2 expression levels.
2.3 Interferon treatment
The PAMs were treated with 1000 IU/ml of IFN-beta (Peprotech, Rocky Hill, NJ, USA) for the times indicated (i.e., 0, 6, 12, 24, 36, and 48), after which the cells were collected and subjected to qRT-PCR to analyze the OAS2 expression levels.
2.4 Plasmid construction The porcine OAS2 gene was amplified from cDNA from the PAMs using the following primer sequences:
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2.5 RNA interference Nonsense siRNA, OAS2 siRNA, retinoic acid inducible gene I (RIG-1) siRNA and RNase L siRNA were designed and synthesized by GenePharma Co., Ltd (Suzhou, China).
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2.6 Cell transfection and viral infections
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CRL-2843-CD163 cells were maintained in 24-well plates and, when the cell monolayer reached 60-70% confluence, 800 ng of the pFLAG-pOAS2 expression vector or the control expression vector (p3xFLAG-CMV -7.1) were transfected into the cells using lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. After 24 h, the cells were infected with PRRSV BJ-4 (MOI=1) for another 24 h, and
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the mRNA levels of PRRSV N (ORF7), OAS2, interferon regulatory factor (IRF)3, IRF7, Toll-like receptor (TLR)3, TLR7, RIG-1, melanoma differentiation-associated protein 5 (MDA5), myeloid differentiation primary response 88 (MyD88), TANK binding kinase 1 (TBK1), nuclear factor-κB (NF-κB), interleukin (IL)-8, IL-1
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western blotting.
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beta, IFN-alpha, and IFN-beta mRNAs were detected by qRT-PCR. IRF3 and pIRF3 proteins were detected by
PAMs were transfected with 60 nM of OAS2 siRNA, RNase L siRNA, RIG-1 siRNA or nonsense siRNA, and then infected with PRRSV BJ-4 (MOI=1) for another 24 h, and PRRSV N mRNA was detected by qRT-PCR.
CRL-2843-CD163 cells were transfected with 60 nM of RNase L siRNA, RIG-1 siRNA or nonsense siRNA and, the CRL-2843-CD163 cells, which RNase L or RIG-1 were knockdown, were transfected with pFLAG-pOAS2 or the negative control vector, and infected with BJ-4 (MOI=1) for another 24 h, after which
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2.7 Cell viability assays The enhanced Cell Counting Kit-8 assay (Beyotime Biotech, China) here was used to evaluate cytotoxicity
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levels, whereby pFLAG-pOAS2 or p3xFLAG-CMV TM-7.1 expression vectors (800 ng each) were transfected individually into CRL-2843-CD163 cells plated in 24-well plates, and the cytotoxicity of OAS2 was evaluated. PAMs plated in 24-well plates were transfected with 60 nM of OAS2 siRNA, RNase L siRNA, RIG-1
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siRNA or nonsense siRNA for 48 h, the cytotoxicity was evaluated as stated above.
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2.8 Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA from the cells was treated with TRIzol (Invitrogen, USA). Reverse transcriptase reactions were performed using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Dalian, China), while SYBR Ex Taq™ (Takara, Dalian, China) was used in the polymerase chain reactions, and the machine was the 7500 Fast
2.9 Western blotting
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Real-time PCR System (Applied Biosystems, Foster city, CA).
CRL-2843-CD163 cells were seeded in 24-well plates and then transfected with the pFLAG-OAS2
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expression vector, and then infected with PRRSV BJ-4 (MOI=1). 48 h later, western blotting was performed as
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described previously [25] using either an anti-Flag monoclonal antibody (1:200, Abnova, Taipei, Taiwan) , an anti-GAPDH monoclonal antibody (1:1000, Beijing Biosynthesis Biotech Co., Ltd, China), an anti-IRF3 monoclonal antibody (1:1000, Proteintech Group, China), or an anti-pIRF3 monoclonal antibody (1:1000, Cell Signaling Technology, USA). The membranes were washed three times with Tris-buffered saline containing Tween-20 (TBST), and then reacted with
horseradish peroxide-conjugated goat anti-rabbit IgG (1:10000,
Zhongshan Golden bridge Biotechnology, China) or horseradish peroxide-conjugated goat anti-mouse IgG (1:10000, Jackson ImmunoResearch, West Grove, PA, USA) for one hour at 37℃, and then washed with TBST
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2.10 Viral titers
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Supernatants from the transfected groups were obtained at 48 h post infection, and PRRSV titers were measured.
2.11 Luciferase reporter assays
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CRL-2843-CD163 cells were seed in 24 well-plates, when they reached 70% confluence, 200 ng of
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pIFN-Beta-Luc (luciferase reporter plasmid), 80 ng of pRL-TK, and the expression vector (500 ng) or the control vector were transfected into the cells with lipofectamine 2000 (Invitrogen, USA). 24 h later, these cells were transfected with 1.5 µg Poly (I:C) for 9 h, Luciferase Reporter System (Promega, Madison, WI, USA) was used to detect the luciferase activity, and the CRL-2843-CD163 cells were also
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infected with PRRSV for 24 hours(MOI=1), and luciferase reporter assays were performed.
2.12 Secreted IFN-beta protein measurements
procedures.
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The secreted IFN-beta was detected with an ELISA kit (Cloud-Clone Group, China) by the manufacturer’s
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2.13 Statistical analysis
Every experiment has three independent experiments; each independent experiment has three technical replicates. GraphPad Prism 5 was used to analyze the results using Student's t-test.
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ACCEPTED MANUSCRIPT Results 3.1 IFN-beta and PRRSV induce porcine OAS2 mRNA expression in PAMs The results showed that from 6 hours post treatment with 1000 IU/ml IFN-beta, porcine OAS2 mRNA
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expression level increased by 40-fold in PAMs (Fig. 1A). The peak mRNA expression level for the OAS2 gene occurred at 12 h post treatment in the PAMs.
The expression kinetics of OAS2 in the PAMs stimulated by PRRSV was also investigated. 24 h later, the
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following PRRSV treatment resulted in only an 8-fold increase.
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highest level of OAS expression following PRRSV infection was detected (Fig. 1B). Induction of OAS2 mRNA
3.2 OAS2 overexpression inhibits PRRSV replication in CRL-2843-CD163 cells Immunoblotting revealed that porcine OAS2 was successfully expressed in the CRL-2843-CD163 cells transfected with the pFLAG-pOAS2 expression vector (Fig. 1C). The transfection efficiency of the plasmid (800
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ng) in the 24-well-plate cells was about 25 %, and no significant cytotoxic effect was observed. Relative expression of PRRSV ORF7 mRNA of CRL-2843-CD163 cells transfected with pFLAG-pOAS2 was lower (Fig. 1D). We also found that the TCID50 of the supernatants from cells transfected with
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pFLAG-pOAS2 was lower (Fig. 1E).
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As shown in Fig. 2A, after infection with PRRSV, OAS2 induced a 2.2-fold increase in IFN-alpha mRNA, a 2.0-fold increase in IFN-beta mRNA, a 2.4-fold increase in IRF3 mRNA, a 4.3-fold increase in TLR-3 mRNA, a 4.3-fold increase in RIG-1 mRNA, a 2.3-fold increase in NF-κB mRNA, a 2.6-fold increase in MDA5 mRNA, a 6.3-fold increase in MyD88 mRNA, a 1.9-fold increase in TBK1 mRNA, and no increases in TNF-alpha, IL-8, IL-1beta, IRF7 mRNA levels. In response to PRRSV infection, the supernatant from the CRL-2843-CD163 cells transfected with pFLAG-pOAS2 had a higher concentration of IFN-beta than that of the CRL-2843-CD163 cells transfected with
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ACCEPTED MANUSCRIPT the p3xFLAG-CMV -7.1 vector (Fig. 2C). The active and latent forms of IRF-3 were also analyzed by western blotting, and as shown in Fig. 2D, OAS2 enhanced the phosphorylation level of IRF-3. IFN-beta promoter activity got significantly enhanced by OAS2 induced by Poly (I:C) and PRRSV (Fig. 2E).
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3.3 Knockdown of OAS2 led to increases in PRRSV replication in PAMs Efficiency of OAS2 gene knockdown in PAMs was approximately 70% (Fig. 3A), while PRRSV ORF7 mRNA increased significantly in the OAS2 knockdown group (Fig. 3D). The TCID50 of the supernatants from
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the OAS2 knockdown group was higher (Fig. 3C).
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In siRNA rescue experiments, 60 nM of si-OAS2, in combination with 800 ng of the pFLAG-pOAS2 plasmid were transfected into the CRL-2843-CD163 cells, and 24 h later, cells were infected with PRRSV. The results showed that no increase in the PRRSV ORF7 mRNA expression level was found.
3.4 RNase L- and RIG-1-dependent antiviral activities
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In the PAMs, 60 nM of RNase L siRNA induced a 33% knockdown of RNase L mRNA (Fig. 4A), which in turn induced a 1.9-fold increase in PRRSV ORF7 mRNA (Fig. 4B). In order to detect whether the anti-PRRSV function rely on RNase L, 30 nM of OAS2 siRNA and 30 nM of RNase L siRNA were co-transfected into PAMs,
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followed by infection with PRRSV (MOI=1). In comparison with the control, no increase in PRRSV ORF7
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mRNA expression in the OAS2/RNase L co-silenced group was found (Fig. 4C). For the PAMs, 60 nM of RIG-1 siRNA induced a 30% knockdown of RIG-1 mRNA (Fig.4D), and this facilitated a 1.7-fold increase in PRRSV ORF7 mRNA (Fig. 4E). To determine whether the anti-PRRSV activity was dependent upon the RIG-1 pathway, both of the OAS2 siRNA and RIG-1 siRNA were transfected into PAMs, and then PRRSV infect the cells (MOI=1). We found no increase in PRRSV ORF7 mRNA expression in the OAS2/ RIG-1 co-silencing group (Fig. 4F). In the CRL-2843-CD163 cells, 60 nM of RNase L siRNA induced a 29% knockdown of RNase L mRNA
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ACCEPTED MANUSCRIPT (Fig. 4G), and this induced a 1.8-fold increase in PRRSV ORF7 mRNA (Fig. 4H). To determine whether the anti-PRRSV activity was dependent on the RNase L pathway, CRL-2843-CD163 cells with RNase L knocked-down were transfected with pFLAG-pOAS2 or the negative control vector. No increase in PRRSV
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ORF7 mRNA expression in the RNase L knockdown group was observed (Fig. 4I). In the CRL-2843-CD163 cells, 60 nM of RIG-1 siRNA induced a 28% knockdown of RIG-1 mRNA (Fig.4L), and this facilitated a 1.6-fold increase in PRRSV ORF7 mRNA (Fig. 4M). To determine whether the
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anti-PRRSV activity was dependent on the RIG-1 pathway, CRL-2843-CD163 cells with RIG-1 knocked-down
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were transfected with pFLAG-pOAS2 or the negative control vector. No increase in PRRSV ORF7 mRNA
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expression in the RNase L knockdown group was observed (Fig. 4N).
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ACCEPTED MANUSCRIPT Discussion The current vaccines could hardly inhibit PRRSV infection, there are some factors: high frequency of PRRSV genome mutation, antibody dependent enhancement (ADE) effect, and persistence of virus in the
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infected pigs. So finding new antiviral therapy becomes a useful method to control PRRSV. Up to now, some ISGs are reported to inhibit viruses, for example, IFIT3[26, 27], ISG15[4], Mx2[28], but the effect of OAS were not well-known.
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This paper has shown PRRSV replication was significantly inhibited by overexpression of porcine OAS2,
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but increased following knockdown of porcine OAS2. The antiviral mechanism of OAS is shown in some other researches, but not well understood, overexpression of porcine OAS2 could trigger RIG-I and some other molecules, it has been shown human OASL binds RIG-I and has antiviral activities [29]. Porcine OAS2, which has also been found to have antiviral activities, can also activate the RIG-I pathway, and the innate immune
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character could share sort of similarity for pigs and humans in this respect. During the experiments, we found that some antibodies used for human signaling molecules could not be used in the porcine signaling molecules, maybe some amino acids mutations change the recognition clues for porcine signaling molecules, so this
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become a question for the porcine innate immunes.
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Zheng et al reported that porcine OAS2 can inhibit JEV, via porcine OAS2 functioning through OAS/RNase L signal channel [30], our result consistent with that found for JEV. For the porcine OAS1, we found that overexpressions of OAS1 also inhibit PRRSV, but the effect is not as significant as that of porcine OAS2. Knockdown of OAS1 could increase the replication of PRRSV, and also the effect was not as good as that of porcine OAS2 (date not shown), so we focused on antiviral effect of porcine OAS2 only. We first discovered that porcine OAS2 could induce the phosphorylation of IRF3 to enhance IFN pathway. The host gene could inhibit the virus through this way, there are reports showing that mouse OASL1 could
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ACCEPTED MANUSCRIPT inhibit type I interferon [31], this is different from the porcine OAS, and the resultant innate immune character did not share similarity between pigs and mouse in this respect. Porcine OAS2 has enzymatic and antiviral activities, whether its antiviral activities connect with enzymatic activities needs further research. The porcine
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OAS2 located in the cytoplasm (date not shown), the PRRSV also replicate in the cytoplasm, whether the porcine OAS2 interacted with viral proteins in the cytoplasm needs more research, maybe the PRRSV could
are required for the antivirus activity needs more research.
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escape the innate immunity through inhibiting OAS2 or other OAS, and which critical regions or amino acids
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PRRSV can replicate in the Marc-145 cells which are derived from monkey kidney[32], and in PAMs which are target porcine cells in vivo [33], but PAMs are not easy for plasmid DNA to be transfected, so we used CRL-2843-CD163 cells to investigate the antiviral activity of OAS2 and found that the transfection efficiency of 800 ng of OAS2 in 24-well-plates cells was about 30%. The CRL-2843-CD163 cell line was
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modified from CRL-2843 cells, which involved CD163 transfection into the CRL-2843 cells. Thus, PRRSV has the ability to infect CRL-2843-CD163 cells, a result consistent with that obtained for CRL-2843-CD163 cells and PAMs, which highlights the utility of CRL-2843-CD163 cells for studying the pathogenesis of PRRSV;
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therefore, use of the CRL-2843-CD163 line could sidestep the need for proper preparation of PAMs.
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Given the antiviral activities of OAS2 against RNA viruses [19, 34, 35], it is possible that OAS2 is required for the prevention of RNA viruses [36]. Therefore, it seems that OAS2 is a universal pattern recognition receptor for the defense against invasion with DNA and RNA viruses. In summary, after cloning of porcine OAS2 gene, it was expressed in CRL-2843-CD163 cells. Porcine OAS2 is an inhibitor to PRRSV; it could increase IFN-beta expression. Further studies on the structure and function of porcine OAS2 mediated signaling pathway are needed, maybe these research could help find new ways to control PRRSV infection.
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Acknowledgment: Our work received funds from the National Natural Science Fund (No. 31490601,
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31502040), and the National Program on the Key Basic Research Project (2014CB542702).
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Figure Legends
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Fig. 1 OAS2 overexpression inhibits PRRSV replication (A) PAMs were stimulated by IFN-beta, at different time points (6, 12, 24, 36, and 48); PAMs were subjected to
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qRT-PCR to quantify OAS2 mRNA levels. (B) PRRSV infected the PAMs, at different time points (6, 12, 24, 36, and 48); PAMs were subjected to qRT-PCR to quantify OAS2 mRNA levels. (C) CRL-2843-CD163 cells were transfected with the pFLAG-pOAS2 vector, 48 h later; western blotting was performed to detect expression. (D) pFLAG-pOAS2 or the negative control vectors were transfected into CRL-2843-CD163 cells; 24 h later, PRRSV infected the cells. 48 h later, qRT-PCR was used to measure the PRRSV ORF7 mRNA expression (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (E) TCID50 was used to measure the viral titers (* means a P value was less than 0.05, 17
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Fig. 2 OAS2 enhances RIG-1 signaling
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significant differences. ** means a P value was less than 0.01, very significant differences).
(A) pFLAG-pOAS2 were transfected into CRL-2843-CD163 cells; 24 h later PRRSV infected the cells, 48 h
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later qRT-PCR was used to measure IFN-alpha, IFN-beta, IL-8, IL-1beta, and TNF-alpha mRNA expression levels (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (B) qRT-PCR was performed to measure IFR3, IFR7, TLR3, TLR7, RIG-1, NF-κB,
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MDA5, MyD88, and TBK1 mRNA expression levels (* means a P value was less than 0.05, significant
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differences. ** means a P value was less than 0.01, very significant differences). (C) ELISA was performed to measure IFN-beta protein levels (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (D) Immunoblotting was used to analyze the cells with anti-IRF-3 and anti-pIRF-3 antibodies. (E) OAS2 overexpression enhanced the activity of the IFN-beta promoter. On day 0, the CRL-2843-CD163 cells were cultured. On day 1, 200 ng of pIFN-Beta-Luc, 80 ng of pRL-TK, and the expression vector (500 ng) or the control were transfected into the cells. On day 2, 1.5 µg of Poly (I:C) treated the cells, 9 h later IFN-beta promoter activities were assessed (* means a P value was less than 0.05,
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ACCEPTED MANUSCRIPT significant differences. ** means a P value was less than 0.01, very significant differences). PRRSV also infected cells (MOI=1), and on day 3, luciferase reporter assays were performed, followed by cell harvesting 24 h later (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01,
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very significant differences).
Fig. 3 OAS2 knockdown in PAMs enhances PRRSV replication (A) 60 nM of OAS2 siRNA (si-OAS2) or negative control siRNA (si-NC) were transfected into PAMs, 24 h later, PRRSV infected the cells for 48h (MOI=1). qRT-PCR was used to measure OAS2 expression (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (B) Evaluation of cell cytotoxicity of si-OAS2 in PAMs. (C) Viral titers were investigated via 19
ACCEPTED MANUSCRIPT TCID50 (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (D) qRT-PCR was used to measure PRRSV N expression levels (* means a P value
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was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences).
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Fig. 4 The anti-PRRSV activity of OAS2 is dependent on RNase L and RIG-1
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ACCEPTED MANUSCRIPT (A) PAMs were transfected with 60nM of RNase L siRNA (si- RNase L) or negative control siRNA (si-NC); At 24 h past transfection, PRRSV infected the cells, RNase L expression levels were measured by qRT-PCR (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very
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significant differences). (B) RRSV N mRNA expression levels were measured by qRT-PCR (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (C) 30nM of RNase L siRNA and 30nM of OAS2 siRNA were co-transfected into PAMs; 24 h later, cells were
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infected with PRRSV (MOI=1), RRSV N mRNA expression levels were measured by qRT-PCR. (D) PAMs
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were transfected with 60nM of RNase L siRNA (si- RIG-1) or negative control siRNA (si-Ctrl); 24 h later, cells were infected with PRRSV (MOI=1), RNase L mRNA expression levels were measured by qRT-PCR (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (E) qRT-PCR was used to measure RRSV N mRNA expression levels (* means a P
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value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (F) 30nM of RIG-1 siRNA and 30nM of OAS2 siRNA were co-transfected into PAMs; 24 h later, cells were infected with PRRSV (MOI=1), RRSV N mRNA expression levels were measured by qRT-PCR.
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(G) CRL-2843-CD163 cells were transfected with 60nM of RNase L siRNA (si- RNase L) or negative control
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siRNA (si-NC); 24 h later, PRRSV was inoculated at a MOI of 1 for 48 h. qRT-PCR was used to measure RNase L expression levels (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (H) qRT-PCR was used to measure RRSV N mRNA expression levels (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (I) CRL-2843-CD163 cells were transfected with 60nM of RNase L siRNA; 24 h later, the CRL-2843-CD163 cells with RNase L knocked down were transfected with 400 ng of pFLAG-pOAS2 or the negative control vector, and cells were infected with PRRSV, qRT-PCR was used to measure RRSV N mRNA
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ACCEPTED MANUSCRIPT expression levels. (L) CRL-2843-CD163 cells were transfected with 60nM of RNase L siRNA (si- RNase L) or negative control siRNA (si-NC); 24 h later, cells were infected with PRRSV. qRT-PCR was used to measure RNase L expression (* means a P value was less than 0.05, significant differences. ** means a P value was less
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than 0.01, very significant differences). (M) qRT-PCR was used to measure RRSV N mRNA expression levels (* means a P value was less than 0.05, significant differences. ** means a P value was less than 0.01, very significant differences). (N) CRL-2843-CD163 cells were transfected with 60nM of RIG-1 siRNA,24 h later, the
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CRL-2843-CD163 cells with RIG-1 knocked down were transfected with 400 ng of pFLAG-pOAS2 or the
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negative control vector, and cells were infected with PRRSV, qRT-PCR was used to measure RRSV N mRNA
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ACCEPTED MANUSCRIPT Table1 Primers used in the research primer sequence TATGAATTCAATGGGAAACTGGGGGTCCC
OAS2- R
CGCGACGACTCAGTCCATGAATCTCC
qOAS2-F
ATGGTGTGAACGCAAACTGA
qOAS2-R
AACCTTCAGCCGTGTCAAAG
qGAPDH-F
CTGCCGCCTGGAGAAACCT
qGAPDH-R
GCTGTAGCCAAATTCATTGTCG
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OAS2- F
qIRF3-F
CCACCAAACUGAAGGAUUU AAAUCCUUCAGUUUGGUGG GCCCUUAACCAAGCAGGUUTT AACCUGCUUGGUUAAGGGCTT UGGAAGAGAUGAAUGCAUA UAUGCAUUCAUCUCUUCCA AAGGTTGTCCCCATGTGTCTCCG
qIRF3-R
GGAAATGTGCAGGTCCACCGTG
qIRF7-F
TCCAGCCGAGATGCTAAGTG
qIRF7-R
GTCCAAGTCCTGCCCGATGT
qORF7-F
AAACCAGTCCAGAGGCAAGG
qORF7-R
GCAAACTAAACTCCACAGTGTAA
qIFN-beta -F
CTAGCACTGGCTGGAATGAGACT
Si-OAS2
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Si-RIG-1
qIFN-beta -R
GGCCTTCAGGTAATGCAGAATC
qTNF-alpha-F
qIL-8-R
ACGGGCTTATCTGAGGTTTGAG
GGCAGTTTTCCTGCTTTCT CAGTGGGGTCCACTCTCAAT
CAGAGCAGCGGCGGAATC
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qRIG-1-F
CACCACGCTCTTCTGCCTAC
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qTNF-alpha-R qIL-8-F
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Si-RNase L
qRIG-1-R
ACTCAAGGTTGCCCAT
qTLR7-F
GAACTGTTTC TCTACAACA
qTLR7-R
AGACTTGTAATTCTGTCA
qTLR3-F
TACTGTACAC AACTTCTACC
qTLR3-R
TTAAATCCTCCATCCAAGG
qNF-κB-F
CCAGCACCTCCACTCCATTC
qNF-κB-R
ACATCAGCACCCAAAGACACC
qMDA5-F
CGAATTAACAGGCACCGATT
qMDA5-R
CGTCCAGACTTGGCTGATCT
qMyD88-F
CTCCGGAGCG GAGTCCGCG
ACCEPTED MANUSCRIPT GCCAGCCCAGTCCAGTCC
qTBK1-F
CCAGTGGAT GTTCAAAT
qTBK1-R
CTCCCACATGGACAAAAT
AC C
EP
TE D
M AN U
SC
RI PT
qMyD88-R
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Highlights We firstly found that porcine 2’, 5’-oligoadenylate synthetase (OAS2) could inhibit the PRRSV replication, and the inhibition was dependent by RNase L. Porcine OAS2 suppressed replication of PRRSV in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. Co-silencing of RNase L and OAS2 revealed that the anti-PRRSV function of porcine OAS2 was RNase L dependent. Our findings show a mechanism by which porcine OAS2 contributes to host anti-PRRSV responses by enhancing RIG-I activation.