2.1. Cell lines and the virus
Porcine alveolar macrophages (PAMs), isolated from lung lavage samples of 7-week-old pigs which were brought from Henan academy of agricultural sciences, free of PRRSV, pseudorabies virus, porcine circovirus type 2, and classical swine fever virus (CSFV), were cultured in the Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA) at 37 ℃, in a humidified atmosphere containing 5% CO2.
CRL-2843-CD163, a stable porcine macrophage cell line that can be infected by PRRSV, was kindly provided by Prof. Enmin Zhou (Northwest A&F University, Lingyang, China) [35]. This cell line was grown in RPMI 1640 medium supplemented with 6% of FBS (Sijiqing, ZhejiangTianhang Biotechnology Co. Ltd., China) at 37 ℃, in a humidified atmosphere containing 5% CO2.
Marc-145 cells (a monkey kidney cell line, ATCC catalogue numbers CRL-12231) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS (Sijiqing, ZhejiangTianhang Biotechnology Co. Ltd., China) at 37 ℃, in a humidified atmosphere containing 5% CO2.
The type 2 PRRSV BJ-4 strain (GenBank accession No. AF331831) was kindly provided by Prof. Hanchun Yang (China Agricultural University, Beijing, China).
2.2. Expression of OASL during PRRSV infection of PAMs
PAMs were infected with PRRSV strain BJ-4 at a multiplicity of infection (MOI) of 1.0 for different periods (0, 6, 12, 24, 36, or 48 h). The cells were then processed for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of OASL mRNA expression.
2.3. Expression of OASL after stimulation of PAMs with IFN
PAMs were stimulated with human IFN-β (PeproTech) which was diluted with PBS at a concentration of 1000 IU/mL for different periods (0, 6, 12, 24, 36, or 48 h). The cells were then subjected to treatment for qRT-PCR determination of OASL mRNA expression.
2.4. Molecular cloning
Porcine OASL (GenBank accession No. NM_001031790.1) was cloned from the complementary DNA (cDNA) extracted from PAMs, using the following primer sequences: 5′-CCGGAATTCTGGAGCTATTTTACACCCCAGC-3′ (OASL-For) and 5′-AAGGAAAAAAGCGGCCGCTCAGTCACAGCCTTTGGCTGAGA-3′ (OASL-Rev). After double digestion, the purified product was ligated with the p3xFLAG-CMV™-7.1 vector (Sigma, USA) to generate the pCMV-3xFLAG-7.1-OASL expression plasmid with the Mix & Go! E. coli Transformation Kit and ZymoPURE Ⅱ Plasmid Midiprep Kit (ZYMO RESEARCH, USA).
2.5. Small interfering RNA (siRNA) synthesis
siRNAs were employed to identify the genes or proteins involved in the antiviral mechanism of OASL. The control siRNA (si-Ctrl, a scrambled control siRNA), OASL siRNA (si-OASL), RIG-I siRNA (si-RIG-I), RNase L siRNA (si-RNase L), and melanoma differentiation-associated protein 5 (MDA5) siRNA (si-MDA5) were all ordered from GenePharma Co., Ltd. (Suzhou, China). The siRNA sequences can be found in Table 1.
Table 1
Primers used in the research
primer | sequences |
OASL- For | CCGGAATTCTGGAGCTATTTTACACCCCAGC |
OASL- Rev | AAGGAAAAAAGCGGCCGCTCAGTCACAGCCTTTGGCTGAGA |
qOASL-F | CTGGTGGCATTTCTGTGCT |
qOASL-R | AGATGGTGAAGGCGATGG |
qGAPDH-F | CTGCCGCCTGGAGAAACCT |
qGAPDH-R | GCTGTAGCCAAATTCATTGTCG |
qIRF3-F | AAGGTTGTCCCCATGTGTCTCCG |
qIRF3-R | GGAAATGTGCAGGTCCACCGTG |
qIRF7-F | TCCAGCCGAGATGCTAAGTG |
qIRF7-R | GTCCAAGTCCTGCCCGATGT |
qN-F | AAACCAGTCCAGAGGCAAGG |
qN-R | GCAAACTAAACTCCACAGTGTAA |
qIFN-beta -F | CTAGCACTGGCTGGAATGAGACT |
qIFN-beta -R | GGCCTTCAGGTAATGCAGAATC |
qTNF-alpha-F | CACCACGCTCTTCTGCCTAC |
qTNF-alpha-R | ACGGGCTTATCTGAGGTTTGAG |
qIL-8-F | GGCAGTTTTCCTGCTTTCT |
qIL-8-R | CAGTGGGGTCCACTCTCAAT |
qRIG-I-F | CAGAGCAGCGGCGGAATC |
qRIG-I-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 |
qMyD88-R | GCCAGCCCAGTCCAGTCC |
qTBK1-F | CCAGTGGAT GTTCAAAT |
qTBK1-R | CTCCCACATGGACAAAAT |
Si-OASL | GGCACAUGAGCGUUUCCAUTT |
Si-RIG-I | GCAGGUUAUUCUGGACUUUTT |
Si-MDA-5 | CCUCAGAUAUUGGGACUAATT |
Si-RNase L | UGGAAGAGAUGAAUGCAUATT |
2.6. Transfection and infection
CRL-2843-CD163 cells were transfected with 800 ng of the pCMV-3xFLAG-7.1-OASL expression plasmid or control expression vector (pCMV-3xFLAG-7.1) by means of Lipofectamine 2000 (Life Technologies).
After 24 h of incubation, the cells were infected with PRRSV (at MOI of 0.1 or 1.0) for 24 h. qRT-PCR was then carried out to determine the mRNA expression of various factors, such as PRRSV nucleoprotein (N, ORF7), OASL, RIG-I, and MDA5, as well as the cytokines IFN-β, IFN-α, and pro-interleukin 1 beta (pro-IL-1β).
Total IRF3 and phosphorylated IRF3 (p-IRF3) proteins were quantified by western blot analysis.
For the siRNA experiments, PAMs were transfected with 60 nM si-OASL, si-RNase L, si-RIG-I, si-MDA5, or si-Ctrl via the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen). At 24 h post-transfection, the cells were infected with PRRSV (MOI 1.0) for another 24 h, mRNA expression of the respective genes was determined by qRT-PCR, and the viral titer was tested by TCID50.
For the siRNA and plasmid cotransfection experiments, CRL-2843-CD163 cells were transfected with 60 nM si-RNase L, si-RIG-I, si-MDA5, or si-Ctrl and 800 ng plasmid via the Lipofectamine 2000 Transfection Reagent (Invitrogen). At 24 h post-transfection, the cells were infected with PRRSV (MOI 1.0) for another 24 h, mRNA expression of the respective genes was determined by qRT-PCR, and the viral titer was tested by TCID50.
2.7. qRT-PCR
Total-RNA samples from PAMs and CRL-2843-CD163 cells were extracted with the TRIzol Reagent (Life Technologies) and then subjected to reverse-transcriptase treatment by means of the First Strand cDNA Synthesis Kit (Takara, Dalian, China) and qPCR. qPCR was carried out on a 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA) with the primers listed in Table 1. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was analyzed as an internal control, and relative changes in the expression of the target genes were calculated by the 2−△△Ct method [36].
2.8. Cell survival experiments
The toxicity of porcine OASL and various siRNAs toward PAMs and CRL-2843-CD163 cells was tested with the Enhanced Cell Counting Kit-8 (Solarbio, Beijing, China).
2.9. Western blot analysis
Western blotting was carried out as described previously [37–39]. The primary antibodies were as follows: an anti-FLAG monoclonal antibody (1:200; Abnova, Taipei, Taiwan), anti-GAPDH antibody (1:500; Beijing Biosynthesis Biotech Co., Ltd., Beijing, China), anti-IRF3 antibody (1:1000, Proteintech Group, China), anti-p-IRF3 antibody (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-OASL antibody (1:1000; Abcam, USA), anti-RNase L antibody (1:200; Santa Cruz, USA), anti-RIG-I antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA), and anti-MDA-5 antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies were a horseradish peroxide-conjugated goat anti-rabbit IgG antibody (1:10000, Zhongshan Golden Bridge Biotechnology, China), a horseradish peroxide-conjugated goat anti-mouse IgG antibody (1:10000, Jackson ImmunoResearch, West Grove, PA, USA), or a horseradish peroxide-conjugated goat anti-chicken IgY antibody (1:10000, Abbkine, USA).
2.10. Luciferase reporter assay
CRL-2843-CD163 cells were transfected with 100 ng of pIFN-beta-Luc, 20 ng of pRL-TK, and 400 ng of the expression vector or control vector by means of Lipofectamine 3000 (Life Technologies). After 12 h, 1.5 µg of poly (I: C) (InvivoGen, Paris, France) was transfected into the cells for 9 h. The cells were then subjected to a luciferase reporter assay (Promega, Madison, WI, USA) to determine the IFN-β promoter reporter activity. In another experiment, CRL-2843-CD163 cells transfected as described above were infected with PRRSV (MOI 1.0) for 24 h, and the luciferase reporter assay was then conducted.
2.11. Virus titers
Marc-145 cells were used to determine the PRRSV titers in the supernatants of cultures treated with different vectors and siRNAs. The tissue culture infectious dose 50 (TCID50) was determined.
2.12. Statistical analysis
All experiments were biologically repeated three times, data represent means ± standard deviations of three independent experiments, and all assays were conducted in triplicate. The results were analyzed by the Student’s t- test. Data with a P value < 0.05 were considered statistically significant.