2.1. Cells and Viruses
Porcine alveolar macrophages (PAMs), isolated from lung lavage samples of seven-week old pigs which were free of PRRSV, pseudorabies virus, porcine circovirus type 2, and classical swine fever virus, were cultured in the Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Life Technologies) at 37 ℃, in a humidified atmosphere containing 5% CO2.
CRL-2843-CD163, a stable porcine macrophage cell line that could be infected by PRRSV, was kindly provided by Prof. Enmin Zhou (Northwest A&F University, Lingyang, China) . This cell line was grown in RPMI 1640 medium (Life Technologies) supplemented with 6% of fetal bovine serum (Sijiqing, ZhejiangTianhang Biotechnology Co. Ltd., China) at 37 ℃, in a humidified atmosphere containing 5% CO2.
Marc-145 cells (American Type Culture collection, Manassas, VA, USA, (ATCC), #CRL-12231) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (Sijiqing, ZhejiangTianhang Biotechnology Co. Ltd., China) at 37 ℃, in a humidified atmosphere containing 5% CO2.
The type II PRRSV BJ-4 strain (GenBank accession no. AF331831) was kindly provided by Prof. Hanchun Yang (China Agricultural University, Beijing, China).
2.2. Expression of pOASL during PRRSV Infection of PAMs
PAMs were infected with PRRSV BJ-4 strain at a multiplicity of infection (MOI) of 1.0 for different times (0, 6, 12, 24, 36, and 48 h). The cells were then processed for reverse-transcription and real-time polymerase chain reaction (RT-PCR) analysis of pOASL mRNA expression and Western blotting for pOASL protein expression.
2.3. Expression of pOASL after Stimulation of PAMs with IFN
PAMs were stimulated with human IFN-β (Pepro-Tech, Rocky Hill, NJ, USA), which was diluted with PBS at a concentration of 1,000 IU/mL for different periods (0, 6, 12, 24, 36, and 48 h). The cells were then subjected for RT-PCR analysis of pOASL mRNA expression and Western blotting for pOASL protein expression.
2.4. Molecular Cloning
pOASL (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 products were ligated with the p3xFLAG-CMV™-7.1 vector (Sigma-Aldrich, St. louis, MO, USA, #E7533) to generate the pCMV-3xFLAG-7.1-OASL expression plasmid. Mix & Go! E. coli Transformation Kit and Zymopure Plasmid Midiprep Kit (Zymo Research, Irvine, CA, USA) were used for cloning and plasmid construct.
2.5. Small Interfering RNA (siRNA) Synthesis
SiRNAs were used to identify the genes or proteins involved in the antiviral mechanism of pOASL. The nontargeting control siRNA (si-NC), 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 were listed in Table 1.
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) via Lipofectamine 2000 Transfection Reagent (Life Technologies). After 24 h of incubation, the cells were infected with PRRSV (MOI of 0.1 and 1.0) for 24 h. RT-PCR was then carried out to determine the mRNA expression levels of various factors.
For the siRNA transfection experiments, CRL-2843-CD163 cells were transfected with 60 nM si-OASL, si-RNase L, si-RIG-I, si-MDA5, or si-NC via the Lipofectamine RNAiMAX Transfection Reagent (Life Technologies). At 24 h post-transfection, the cells were infected with PRRSV (MOI 1.0) for 24 h, PRRSV genomic copy number in the supernatant was determined by RT-PCR, and PRRSV titers were expressed as TCID50.
For the siRNA and plasmid co-transfection experiments, CRL-2843-CD163 cells were transfected with 60 nM si-RNase L, si-RIG-I, si-MDA5, or si-NC and 800 ng pCMV-3xFLAG-7.1-OASL expression plasmid via the Lipofectamine 2000 Transfection Reagent (Life Technologies). At 24 h post-transfection, the cells were infected with PRRSV (MOI 1.0) for 24 h, PRRSV genomic copy number in the supernatant was determined by RT-PCR, and PRRSV titers were expressed as TCID50.
Total RNA 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). RT-PCR 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 .
2.8. Cell Survival Experiments
The toxicity of pOASL 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 Blotting and Immunoprecipitation
Western blotting and Immunoprecipitation were carried out as described previously [39-42]. The primary antibodies were as follows: anti-FLAG monoclonal antibody (Bioss antibodies, Beijing, China, #bs-0879R), anti- Anti-β-actin Polyclonal Antibody (Solarbio, # K006153P), anti-GAPDH antibody (Solarbio, # K106389P), anti-OASL antibody (Abcam, Cambridge, MA. USA, # ab155422), anti-RNase L antibody (Santa Cruz Biotechnology, Dallas, TX, USA, # sc-74405), anti-RIG-I antibody (Cell Signaling Technology, Danvers, MA, USA, #3743), and anti-MDA5 antibody (Cell Signaling Technology, #5321). Secondary antibodies were horseradish peroxide-conjugated rabbit anti-mouse IgG antibody (Santa Cruz Biotechnology, #sc-358914), and mouse anti-rabbit IgG antibody (Santa Cruz Biotechnology, # sc-2357).
2.10. Luciferase Reporter Assay
CRL-2843-CD163 cells were transfected with 200 ng of reporter plasmid, 20 ng of pRL-TK, and 400 ng of the flag tagged OASL plasmid via the Lipofectamine 3000 Transfection Reagent (Life Technologies). After 24 h, 1.5 µg of poly (I: C) (InvivoGen, San Diego, CA, USA) treat the cells for 9 h. The cells were then subjected to luciferase reporter assay system (Promega, Madison, WI, USA) to test the promoter activity.
2.11. Virus Titers
Marc-145 cells were used to determine the PRRSV titers in the supernatants. PRRSV titers were expressed as TCID50.
2.12. Statistical Analyses
All experiments were repeated three times, data were analyzed by Student’s t-test. Differences were considered statistically significant when values of p < 0.05.
The sample size was sufficient for the data analysis using paired two-tailed Student’s t-test. For all Statistical analyses, the differences were considered to be statistically significant at values of p < 0.05.