Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

Key Points

• PRRSV infection induces SOCS1 upregulation.

• PRRSV N protein upregulates SOCS1, and NLS-2 is essential for this function.

• AP-1 signaling pathways are indispensable for PRRSV-induced SOCS1 expression.

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous economic losses in the swine industry since its emergence in the late 1980s. PRRSV exploits various strategies to evade immune responses and establish chronic persistent infections. Suppressor of cytokine signaling (SOCS) 1, a member of the SOCS family, is a crucial intracellular negative regulator of innate immunity. In this study, it was shown that SOCS1 can be co-opted by PRRSV to evade host immune responses, facilitating viral replication. It was observed that PRRSV induced SOCS1 production in porcine alveolar macrophages, monkey-derived Marc-145 cells, and porcine-derived CRL2843-CD163 cells. SOCS1 inhibited the expression of IFN-β and IFN-stimulated genes, thereby markedly enhancing PRRSV replication. It was observed that the PRRSV N protein has the ability to upregulate SOCS1 production and that nuclear localization signal–2 (NLS-2) is essential for SOCS1 induction. Moreover, SOCS1 upregulation was dependent on p38/AP-1 and JNK/AP-1 signaling pathways rather than classical type I IFN signaling pathways. In summary, to our knowledge, the findings of this study uncovered the molecular mechanism that underlay SOCS1 induction during PRRSV infection, providing new insights into viral immune evasion and persistent infection.

Introduction

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failure in sows and respiratory distress in pigs of all ages. Since it was first reported in 1987, PRRS has remained one of the most important viral diseases of pigs, causing immense economic losses in the pig industry worldwide (1, 2). The etiologic agent of PRRS is PRRS virus (PRRSV), an enveloped, nonsegmented, single-stranded, positive-sense RNA virus belonging to the genus Rodartevirus, the family Arteriviridae, and the order Nidovirales (3). The PRRSV genome is ∼15.4 kb in length and contains at least 11 open reading frames (ORFs): ORF1a, ORF1b, ORF2a, ORF2b, ORFs3–7, ORF5a, and ORF2TF. ORF1a and ORF1b constitute ∼75% of the genome in the 5′ region and encode at least 16 nonstructural proteins (nsps), including nsp1α, nsp1β, nsp2–6, nsp2TF, nsp2N, nsp7α, nsp7β, and nsp8–12 (4–6). ORF2a, ORF2b, ORFs3–7, and ORF5a encode eight structural proteins, including two major membrane proteins (GP5 and M), five minor membrane proteins (GP2a, GP3, GP4, GP5a, and E), and the nucleocapsid (N) protein. PRRSV evolves quickly and is characterized by extensive genetic variation, mutation, and recombination, making the prevention and control very challenging.

The typical immune features of PRRSV infection contain weak and aberrant innate immune responses, delayed appearance of neutralizing Abs, and inefficient cellular immune responses (7–9). PRRSV employs numerous mechanisms to establish persistent infection. For example, PRRSV interferes with the induction and function of IFN by using an N protein or nsps (10, 11). PRRSV blocks nuclear translocation of STAT 1 and STAT2, leading to the inhibition of IFN-activated JAK-STAT signaling (12). Nonetheless, the mechanisms of immune evasion by PRRSV have not been fully characterized. Little is known with respect to the effects of PRRSV infection on negative regulators of host immune response, such as suppressor of cytokine signaling (SOCS) family proteins.

Intracellular SOCS proteins are involved in manipulating both innate and adaptive immunity, including negative regulation of the JAK/STAT and TLR signaling cascades, dendritic cell activation, T cell differentiation, and Th cell regulation (13–16). There are eight members of the SOCS family (SOCS1-SOCS7 and CIS), each of which mainly contains a central SRC homology 2 (SH2) domain, a conserved C-terminal SOCS-box domain, and an N-terminal domain with variable length (17, 18). The central SH2 domain determines the target of SOCS proteins via binding to phosphorylated tyrosine residues on activated cytokine receptors. The SOCS-box interacts with elongins B and C, Cullin5, and RING-box-2 to form an E3 ubiquitin ligase complex, mediating the degradation of associated proteins (19–22). Among the SOCS proteins, SOCS1 and SOCS3 are best characterized. In addition to ubiquitin-mediated proteasomal degradation, SOCS1 and SOCS3 can inhibit tyrosine kinase activity directly because they contain a unique kinase inhibitory region that is proposed to function as a pseudosubstrate to interact with the JAK activation loop (23). Therefore, SOCS1 and SOCS3 are significant inhibitors of the JAK-STAT pathways. Recent studies have also shown that SOCS1 and SOCS3 strongly suppressed TLR7-mediated type I IFN production (24, 25).

As key physiological regulators of the immune system, SOCS protein expression is tightly regulated in a cell type– and stimulus-specific manner (26, 27). SOCS expression is classically induced by cytokines, especially type I IFNs. Previous studies indicated that SOCS proteins are also induced by various stimuli, such as cAMP, LPS, TNF-α, isoproterenol, and statins through different pathways (28–31). Several viruses have been demonstrated to induce SOCS protein expression and exploit their functions to evade host immune defenses and promote replication (32–39). For example, HIV type 1 (HIV-1) infection induces SOCS1 expression, which facilitates HIV-1 replication by directly binding to the matrix and N regions of the HIV-1 p55 Gag polyprotein to enhance its stability and trafficking (40). Okumura et al. (38) demonstrated that Ebola virus–like particles stimulate the induction of SOCS3, leading to enhanced ubiquitinylation and budding of Ebola virions. Recent studies have shown that some viruses, like Japanese encephalitis virus and transmissible gastroenteritis virus, employ microRNAs to manipulate SOCS protein expression, thus evading cellular antiviral responses for promoting virus replication (41, 42). Thus, it can be seen that viruses hijack the host SOCS system through different mechanisms to influence viral replication, pathogenesis, and immune evasion.

The relationships between SOCS proteins and PRRSV infection have never been elucidated. Therefore, it was investigated as to whether SOCS proteins played a role in PRRSV immune evasion, thereby contributing to persistent infection. It was also assessed as to whether SOCS1 affected PRRSV replication as well as the underlying mechanism of SOCS production. As expected, it was observed that SOCS1 inhibited IFN and IFN-stimulated genes (ISGs) and facilitated PRRSV replication. PRRSV-induced SOCS1 production was not due to classical IFN signaling but required p38/AP-1 and JNK/AP-1 signaling pathways. In addition, the PRRSV N protein was involved in SOCS1 upregulation. Using deletion analysis, it was identified that the nuclear localization signal (NLS)–2 of PRRSV N protein was essential for SOCS1 activation.

Materials and Methods

Cells and viruses

Marc-145 cells were maintained in DMEM supplemented with 10% FBS (Life Technologies), 100 U/ml penicillin, and 100 μg/ml streptomycin. CRL2843-CD163 cells (kindly provided by professor E.-m. Zhou from the College of Veterinary Medicine, Northwest A&F University) were a cell line stably expressing CD163 in CRL2843 and maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (43). Primary porcine alveolar macrophages (AMs) were obtained from lung lavage of 4- to 6-wk-old specific pathogen-free piglets, as described previously (44), and were stored in liquid nitrogen until they were used. Porcine AMs were revived and cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. All the cultures were performed at 37°C under a humidified atmosphere containing 5% CO₂.

The highly pathogenic PRRSV strain HN07-1 (GenBank identifier [ID]: KX766378.1, https://www.ncbi.nlm.nih.gov/nuccore/KX766378.1) and the NADC30-like strain HNhx (GenBank ID: KX766379, https://www.ncbi.nlm.nih.gov/nuccore/KX766379) were previously isolated by our laboratory in 2007 and 2016, respectively, in China (45–47). The low-pathogenic PRRSV strain BJ-4 (GenBank ID: AF331831, https://www.ncbi.nlm.nih.gov/nuccore/AF331831) and PRRSV strain GZ11-G1 (GenBank ID: KF001144, https://www.ncbi.nlm.nih.gov/nuccore/KF001144) were kind gifts from Prof. H. Yang (China Agricultural University) (48). The strains HN07-1, HNhx, and BJ-4 belong to PRRSV-2 species. The strain GZ11-G1 belongs to PRRSV-1 species. The heat-inactivated HN07-1 (Heat-HN07-1) was generated by incubating the strain HN07-1 in a water bath at 65°C for 1 h. The UV-inactivated HN07-1 (UV-HN07-1) was obtained by irradiation strain HN07-1 with 254 nm of UV light for 2 h. Viruses were titrated using the Reed-Muench method and expressed as tissue culture ID₅₀ (TCID₅₀). Viruses were stored at −80°C until they were used.

Reagents and Abs

B18R, a vaccinia virus–derived soluble type I IFNR previously reported to have the ability to bind and/or inhibit the activity of type I IFN, was purchased from eBioscience (49, 50). IFN-α and IFN-β were purchased from PBL Assay Science and PeproTech, respectively. Protease inhibitor (cOmplete, EDTA-free, EASYpack of 20) was purchased from Roche. Phosphatase inhibitor (NaF) was purchased from Beyotime. Polyinosinic-polycytidylic acid [poly(I:C)] was purchased from Sigma-Aldrich. HRP-conjugated anti-mouse and anti-rabbit IgG were purchased from Jackson ImmunoResearch.

Plasmids

The codon-optimized porcine SOCS1 gene (sequences provided upon request) was synthesized and subcloned into pEGFP-C1 vector to generate pEGFP-SOCS1 construct by Sangon Biotech. SOCS1 was also amplified using pEGFP-SOCS1 as template and subcloned into pcDNA3.1-Myc/His_A to generate pcDNA3.1-SOCS1-His. Genes that encode PRRSV HN07-1 nsp1α, nsp1β, nsp2, nsp4, nsp5, nsp11, and N protein were amplified by PCR using specific primers (Table I) and cloned into pcDNA3.1-Myc/His_A. The N deletion mutant NΔNLS-2 was amplified by overlap PCR and subcloned into pcDNA3.1-Myc/His_A. Other N deletion mutants (NΔNLS-1, NΔN–N noncovalent interaction domain [N-N], and NΔnucleolar localization signal [NoLS]) were synthesized by Sangon Biotech. All constructs were confirmed by DNA sequencing by Sangon Biotech. The IFN-β–luciferase reporter (IFN-β–Luc) was constructed in our laboratory (51).

Table I. Primers used in cloning in this study.

Name	Sequence (5′-3′)
SOCS1 F	5′-CGAAGCTTATGGTCGCCCACAACCAGGTGG-3′
SOCS1 R	5′-CCGGATCCTCACTTGTCATCGTCGTCCTTGTAGTCG ATCTGGAATG-3′
Nsp4 F	5′-GGGGTACCATGGGCGCTTTCAGAACTCAAAAGC-3′
Nsp4 R	5′-GCTCTAGATTCCAGTTCGGGTTTGGCAGC-3′
Nsp5 F	5′-GGGGTACCATGGGAGGCCTTTCCACAGTTCAAC-3′
Nsp5 R	5′-GCTCTAGACTCGGCAAAGTATCGCAAGAAG-3′
Nsp11 F	5′-CGGGATCCATGGAAGGGTCGAGCTCCCCG-3′
Nsp11 R	5′-CCGCTCGAGTTCAAGTTGAAAATAGGCCGTC-3′
N Fa	5′-GCCCTCGAGATGCCAAATAACAACGGCAAGCAG-3′
N Rb	5′-CGCACCGGTTGCTGAGGGTGAAGCTGTG-3′
N (1–120 nt) Rb	5′-GGGCTTCTCCGGGTTTTTTCCCTTGCCTCTGGACTG-3′
N (142–372 nt) Fa	5′-CAGTCCAGAGGCAAGGGAAAAAACCCGGAGAAGCCC-3′
SOCS1 −2000/100 F	5′-GCGGTACCGTGTCTCCTGCAGTCAGGGTGAAAT-3′
SOCS1 −2000/100 R	5′-GGCAAGCTTCAGTGGGGAGGCATAGCACCTCC-3′
SOCS1 −1359/100 F	5′-GCGGTACCAGAAAGGCTGAGGAGCCCTGG-3′
SOCS1 −381/100 F	5′-GCGGTACCTCCCGGATATAAGTCTCCACTCTGC-3′
SOCS1 −58/100 F	5′-GCGGTACCAGCTATTTATACATCTCCCTCTGCCCAGA-3′

^aThe forward primer of NΔNLS-2 protein deletion mutants.

^bThe reverse primer of NΔNLS-2 protein deletion mutants.

F, forward primer; R, reverse primer.

Genomic DNA was extracted from porcine AMs using TRIzol. A 2.1-kb fragment of the SOCS1 promoter flanking the 5′ region of the SOCS1 gene was amplified by PCR from swine genomic DNA and was cloned between KpnI and HindIII sites of the luciferase reporter vector pGL4.17[luc2/Neo] (pGL4.17-basic) (Promega) to generate the SOCS1 promoter reporter plasmid (−2000/100-Luc). The clone sequences are found at nt 31877143–31879242 of the Sus scrofa genome (GenBank ID: NC_010445.4, https://www.ncbi.nlm.nih.gov/nuccore/NC_010445.4?report=genbank&to=132848913). The SOCS1 promoter–truncated mutants −1359/100-Luc, −381/100-Luc, and −58/100-Luc were generated by PCR using the −2000/100-Luc promoter as template. Element deletion mutants of the SOCS1 promoter, including −308ΔIFN regulatory factor 3 (IRF3)–Luc, −188ΔSp1-Luc, −160ΔSp1-Luc, −116ΔIRF3-Luc, and −104ΔAP-1-Luc, were constructed in pGL4.17-basic by Sangon Biotech. The pRL-TK Renilla luciferase reporter plasmid was used as an endogenous control. All primers used are listed in Table I.

Western blotting

Whole cells were lysed in Radio Immunoprecipitation Assay buffer (Beyotime) supplemented with protease and phosphatase inhibitors. Equal amounts of proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Merck Millipore). The membranes were blocked for 1 h in 5% skimmed milk and incubated with the corresponding primary Abs for 1 h at room temperature. The following primary Abs were used as the manufacturer’s recommended dilution: anti-SOCS1 (3:1000; Cell Signaling Technology [CST]), anti–β-Actin (1:1000; CST), anti-GAPDH (1:1000; CST), anti-His (1:1000; CST), anti–c-Jun (1:1000; Abcam), anti–c-Fos (1:1000; Abcam), anti–phospho-c-Jun (1:1000; Abcam), anti–phospho-c-Fos (1:1000; CST), and anti-N (prepared in our laboratory). The membranes were then incubated with the appropriate secondary Abs for 1 h. Immunoreactive bands were visualized with ECL reagent (CST) according to the manufacturer’s protocols and imaged using a VILBER Fusion FX7.

Overexpression of PRRSV proteins or SOCS1 protein

Marc-145 cells or CRL2843-CD163 cells were transfected with expression vectors that encode PRRSV proteins (nsp1α, nsp1β, nsp2, nsp4, nsp5, nsp11, N protein, or mutants) or SOCS1 using Lipofectamine 2000. Empty vector (pcDNA3.1-Myc/His_A) was used as a control. The cells were lysed for Western blotting using primary anti-His Ab (1:1000; CST), luciferase reporter assays, titer determinations, and quantitative real-time PCR (qRT-PCR).

Assessment of SOCS1 protein levels by ELISA

SOCS1 protein levels in porcine AMs or CRL2834-CD163 cells were measured using a commercially available ELISA kit (catalog number: KS18422; Shanghai KeShun Biological Technology). In brief, porcine AMs were collected from tissue culture dishes and centrifuged at 500 × g at 4°C for 10 min, and they were washed three times with sterile PBS. The final pellets were resuspended in PBS containing 1× protease inhibitor and 1 mM NaF. The cell lysates were subjected to three freeze-thaw cycles and centrifuged at 5000 × g at 4°C for 15 min. The supernatants were subjected to ELISA following the manufacturer’s protocols. The data obtained were expressed as nanograms per microgram of total proteins, as used previously (52).

RNA isolation and qRT-PCR

Total RNA was isolated from porcine AMs, CRL2843-CD163 cells, or Marc-145 cells using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. cDNA was prepared from total RNA by reverse transcription using PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio). RT-PCR was performed with a FastStart Universal SYBR Green Master (Rox) Kit (Roche) on a 7500 fast real-time PCR system (Applied Biosystems). Fold changes in gene expression relative to controls was calculated using the 2^−ΔΔCT method (53). GAPDH was set up as an endogenous control. Gene-specific primers are listed in Table II.

Table II. Quantitative RT-PCR primers used in this study.

Genes	Sequence (5′-3′)
Porcine GAPDH F	5′-CCTTCCGTGTCCCTACTGCCAAC-3′
Porcine GAPDH R	5′-GACGCCTGCTTCACCACCTTCT-3′
Porcine SOCS1 F	5′-CGCCCTCAGTGTGAAGATGG-3′
Porcine SOCS1 R	5′-GCTCGAAGAGGCAGTCGAAG-3′
Porcine IFN-β F	5′-TGCAACCACCACAATTCC-3′
Porcine IFN-β R	5′-CTGAGAATGCCGAAGATCTG-3′
Porcine IFN-α F	5′-GCCTCCTGCACCAGTTCTACA-3′
Porcine IFN-α R	5′-TGCATGACACAGGCTTCCA-3′
Porcine ISG56 F	5′-TCAGAGGTGAGA AGGCTGGT-3′
Porcine ISG56 R	5′-GCTTCCTGCAAGTGTCCTTC-3′
Porcine OAS1a F	5′-CTGAAGGAGAGGTGCTTCCA-3′
Porcine OAS1a R	5′-GAAGACGACGAGGTCAGCAT-3′
Porcine IFNAR1 F	5′-CCGTGGTATGAGGTTGAG-3′
Porcine IFNAR1 R	5′-TGCTTTATCTTCGGCTTC-3′
Porcine c-Jun (AP-1) F	5′-AGGCGGAGAGGAAGCGTATGAG-3′
Porcine c-Jun (AP-1) R	5′-CTGAGCATGTTGGCGGTGGAC-3′
Porcine c-Fos (AP-1) F	5′-CGAAGGGAAAGGAATAAGATG-3′
Porcine c-Fos (AP-1) R	5′-TCAAGGGAAGCCACAGACA-3′
Monkey GAPDH F	5′-GGGAAGGTGAAGGTCGGAGTCAA-3′
Monkey GAPDH R	5′-TCTCGCTCCTGGAAGATGGTGAT-3′
Monkey SOCS1 F	5′-GTTCCGTTCGCACGCCGATTACC-3′
Monkey SOCS1 R	5′-CACGCTGAGGGCGAAGAAGCAGTT-3′
Monkey ISG56 F	5′-ACGTAACTGAAAATCCACAAGA-3′
Monkey ISG56 R	5′-TGCTCCAGACTATCCTTGACCT-3′
Monkey OAS1a F	5′-GCGAGTTCTCCACCTGCTTCAC-3′
Monkey OAS1a R	5′-ACTAGGCGGATGAGGCTCTTGAG-3′
ORF7 F	5′-AAACCAGTCCAGAGGCAAGG-3′
ORF7 R	5′-GCAAACTAAACTCCACAGTGTAA-3′

F, forward primer; R, reverse primer.

Small interfering RNA knockdown

The specific small interfering RNAs (siRNAs) against SOCS1 (siSOCS1-1: 5′-CCU GCA CGG AGC AUU AAC UTT-3′; siSOCS1-2: 5′-GCC GAC AAU GCA AUC UCC ATT-3′), IFNAR1 (siIFNAR1: 5′-GCC UGG AUG UCA AUA UGU UTT-3′), c-Jun (sic-Jun: 5′-GCA AAG AUG GAA ACG ACC UTT-3′), c-Fos (sic-Fos: 5′-CCU GUC UGG UUC CUU CUA UTT-3′), and a scrambled negative control (NC) RNA (NC: 5′-ACG UGA CAC GUU CGG AGA ATT-3′) were synthesized by GenePharma. The cells were transiently transfected using Lipofectamine RNAiMAX (Invitrogen). Briefly, porcine AMs were transfected with siRNA against SOCS1 (an equimolar mixture of both siRNAs against SOCS1), IFNAR1, c-Jun, c-Fos, or NC. Twenty-four hours later, the cells were infected with PRRSV at a multiplicity of infection (MOI) of 0.1 or 1 for 24 h. Subsequently, qRT-PCR or Western blotting was used to assess the effect of siRNAs on gene expression and other target gene or protein expression.

Luciferase reporter assays

Marc-145 cells or CRL2843-CD163 cells were seeded in 48-well plates and cultured until the cells reached ∼70–80% confluences. The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega). The cells were then infected or mock infected with PRRSV at an MOI of 1 for 24 h. Or the cells were cotransfected with an SOCS1 promoter luciferase reporter plasmid, a pRL-TK Renilla luciferase reporter plasmid, and a plasmid that encodes the indicated PRRSV proteins or empty vector using Lipofectamine LTX and Plus Reagent (Invitrogen). At 24 h postinfection (hpi) or transfection, the cells were lysed, and luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s instructions.

CRL2843-CD163 cells were infected with or without PRRSV at an MOI of 1 for 12 h or transfected with a plasmid that encodes SOCS1 protein or empty vector for 12 h and then transfected with an IFN-β–Luc or pGL4.17-basic and a pRL-TK renilla luciferase reporter plasmid. Twenty-four hours posttransfection, the cells were transfected or mock transfected with poly(I:C) for 12 h. pGL4.17-basic was used as a control of reporter plasmid. The cells were then lysed, and IFN-β luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s instructions.

Inhibition of signal transduction pathways

Porcine AMs were pretreated with the c-Jun inhibitor SR11302, the p38 MAPK inhibitor SB203580, the ERK MAPK inhibitor PD98059, the JNK inhibitor SP600125, or DMSO for 1 h. The cells were then infected with PRRSV at an MOI of 1 for 24 h. Thereafter, the cells were harvested for qRT-PCR or Western blotting analysis.

Marc-145 cells were pretreated with SR11302, SB203580, SP600125, or DMSO for 1 h. Cells were then transfected with a vector that encodes the PRRSV N protein. After 24 h, the cells were harvested for Western blotting. All inhibitors were purchased from Enzo Life Sciences.

Cell viability assay

Cytotoxicity of the soluble type I IFNR B18R and the signaling pathway inhibitors toward porcine AMs or Marc-145 cells were determined using a CellTiter 96 AQ_ueous One Solution Cell Proliferation Assay (catalog no.: G3580; Promega), following the manufacturer’s procedure. Briefly, the cells were incubated with B18R or signaling pathway inhibitors (SR11302, SB203580, PD98059, or SP600125) for 24 h. The medium was replaced with fresh medium containing the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS], and the cells were further incubated at 37°C for 2 h under a humidified atmosphere containing 5% CO₂. The absorbance at 490 nm in each well was recorded using a plate reader. Cytotoxicity was expressed as relative viability by comparison with the number of viable cells in the absence of each compound (set as 100%). Cell survival was calculated by comparison with a standard curve generated from the absorbance at 490 nm of various numbers of cells. The result obtained was expressed as fold changes of reagent-treated cells relative to the survival of mock reagent–treated cells.

Virus titer determination

The cells and supernatants were collected and stored at −80°C. The supernatants were then subjected to three freeze-thaw cycles and centrifuged at 12,000 × g at 4°C for 10 min. The virus titers were determined by TCID₅₀ assay.

Statistical analysis

All experiments were performed with at least three independent replicates. Differences between groups were assessed using two-tailed Student t tests in GraphPad Prism software (version 7.0). A p value < 0.05 was considered significant.

Results

PRRSV infection induces SOCS1 expression in porcine AMs, Marc-145 cells, and CRL2843-CD163 cells

Previous studies have assessed SOCS and CIS transcript expression in various porcine tissues (54). However, little information is available concerning SOCS expression and regulation by PRRSV in porcine AMs as well as several PRRSV-permissive cell lines. To determine whether PRRSV alters the expression of SOCS family of proteins, porcine AMs were infected with PRRSV-2 strain HN07-1 for 24 h, and then SOCS1-7 and CIS mRNA expressions were assessed. SOCS1 was one of the upregulated SOCS proteins during PRRSV infection (data not shown). Thus, the expression kinetics of SOCS1 in several PRRSV-susceptible cells infected with different PRRSV strains were investigated. Porcine AMs were inoculated with PRRSV-2 strain HN07-1 at an MOI of 1. The cells were harvested at the indicated time points to quantify SOCS1 mRNA and protein levels using qRT-PCR, Western blotting, and ELISA. The results show that PRRSV significantly upregulated SOCS1 mRNA levels at 6, 12, 24, and 48 hpi, with expression peaking at 12 hpi (11.5-fold, Fig. 1A). PRRSV infection also increased SOCS1 protein levels at 12, 24, and 36 hpi, with expression peaking at 36 hpi (Fig. 1B, 1C). To confirm this result, porcine AMs were infected with HN07-1 at MOIs of 1, 2, and 10 for 12 h. The results show that the increase of SOCS1 by PRRSV was in a dose-dependent manner (Fig. 1D). Because monkey-derived Marc-145 cells and PRRSV-permissive CRL2843-CD163 cells are typically used to study PRRSV infection in vitro, there was contemplation on the possibility of PRRSV having the same impact on SOCS1 expression in both cell lines. Similar results were obtained in Marc-145 cells and CRL2843-CD163 (Fig. 1E, 1F, Supplemental Fig. 1A, 1B). The effects of the other three representative PRRSV strains, BJ-4 (a PRRSV-2 strain with low pathogenicity), HNhx (an NADC30-like PRRSV-2 strain isolated in China), and GZ11-G1 (a PRRSV-1 strain), were also tested. As shown in Fig. 1G and 1H and Supplemental Fig. 1C and 1D, all strains induced SOCS1 mRNA expression, which suggests that SOCS1 upregulation was not dependent on PRRSV strains. The effects of heat-inactivated and UV-inactivated PRRSV on SOCS1 induction were also investigated. As shown in Fig. 1I, heat-inactivated and UV-inactivated PRRSV hardly induced SOCS1 mRNA expression, which indicates that the induction of SOCS1 by PRRSV depended on virus replication. Taken together, the results of this study demonstrated that PRRSV remarkably upregulated SOCS1 expression at both the transcriptional levels and translational levels (Tables I, II).

FIGURE 1.

PRRSV infection induces SOCS1 expression in porcine AMs and Marc-145 cells. (A–C) Porcine AMs were infected or mock infected with HN07-1 at an MOI of 1. At the indicated time points, the cells were collected to assess SOCS1 mRNA levels (A) using qRT-PCR or SOCS1 protein levels (B) by Western blotting and ELISA (C). For ELISA, data were expressed as picograms per microgram of total protein. (D) Porcine AMs were infected or mock infected with HN07-1 at different MOIs. At 12 hpi, the cells were collected for analysis of SOCS1 mRNA levels using qRT-PCR. (E and F) Marc-145 cells were infected or mock infected with HN07-1 at an MOI of 1. At the indicated time points, the cells were collected, SOCS1 mRNA levels (E) were assessed using qRT-PCR, and SOCS1 protein levels (F) were assessed using Western blotting. (G and H) Porcine AMs were infected or mock infected with HNhx (G) or BJ-4 (H) at an MOI of 1. At the indicated times, the cells were collected, and SOCS1 mRNA levels were assessed using qRT-PCR. (I) Porcine AMs were infected or mock infected with HN07-1, heat-inactivated HN07-1, or UV-inactivated HN07-1. At the indicated time points, the cells were collected, and SOCS1 mRNA levels were assessed using qRT-PCR. All experiments were performed with at least three independent replicates. The results obtained were compared with 0 h (medium control) using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

SOCS1 facilitates PRRSV replication

Based on the fact that SOCS1 is a classical negative feedback regulator of innate immune response, especially the JAK/STAT signaling pathways (55), an investigation was carried out to determine the role of porcine SOCS1 in the regulation of IFN-β production. First, the IFN inhibitory effects of PRRSV were confirmed using IFN-β–Luc assays. As shown in Supplemental Fig. 1E, PRRSV inhibited poly(I:C)-induced IFN-β promoter activities. Next, another investigation was carried out to determine whether SOCS1 had IFN inhibitory effects. CRL2843-CD163 cells were transfected with an empty vector or an IFN-β–Luc plasmid and a pRL-TK Renilla luciferase reporter plasmid in the presence or absence of an SOCS1 expression vector. Transfection with pcDNA3.1-SOCS1-His inhibited IFN-β–Luc activity induced by poly(I:C) (Supplemental Fig. 1F). These results suggested that SOCS1 could inhibit poly(I:C)-triggered IFN-β expression. Also, to investigate whether SOCS1 is involved in the suppression of IFN-β and ISGs upon PRRSV infection, porcine AMs were transfected with siRNAs specific for SOCS1 and then infected with HN07-1. At 24 hpi, the cells were collected, and the mRNA levels of IFN-β and ISGs (ISG56 and OAS1a) were assessed using qRT-PCR. As shown in Supplemental Fig. 1G–I, silencing SOCS1 significantly enhanced IFN-β, ISG56, and OAS1a mRNA expression. Thus, it was speculated that SOCS1 might affect PRRSV replication. PRRSV titers were analyzed after inactivating SOCS1 expression by siRNA in porcine AMs. Porcine AMs were transfected with 20 nM siRNA for 24 h and then infected with HN07-1. At 24 hpi, the cells and supernatants were harvested and analyzed for the detection of PRRSV RNA using RT-PCR and PRRSV titers using a TCID₅₀ assay. As shown in Fig. 2C and 2D, inactivating SOCS1 significantly suppressed PRRSV replication in porcine AMs. The levels of PRRSV ORF7 gene and viral titers were decreased to 34.0 and 16.1%, respectively, after the transfection of siRNA to silent the action of SOCS1. The knockdown efficiency of the siRNA against SOCS1 was assessed by qRT-PCR and ELISA (Fig. 2A, 2B). On the contrary, overexpression of SOCS1 by transfection of Marc145 cells or CRL2843-CD163 cells with SOCS1 expression constructs (pcDNA3.1-SOCS1-His) dramatically enhanced PRRSV replication (Fig. 2E, 2F). Therefore, it was concluded that PRRSV may hijack the host cell SOCS1 to facilitate its own replication.

FIGURE 2.

SOCS1 enhances PRRSV replication. (A–D) Porcine AMs were transfected with 20 nM NC siRNA or siRNAs against SOCS1 (an equimolar mixture of two siRNAs) for 24 h before infecting them with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested, SOCS1 mRNA levels (A) were assessed using qRT-PCR, and SOCS1 protein levels (B) were assessed using ELISA. The cells were collected for PRRSV ORF7 (C) quantification using qRT-PCR or viral enumeration (D) using a TCID₅₀ assay. (E and F) Marc-145 cells (E) and CRL2843-CD163 cells (F) were transfected with pcDNA3.1-Myc/his_A or pcDNA3.1-SOCS1-His and then infected 24 h later with HN07-1 (MOI = 0.1). At 24 hpi, the cells were collected, and SOCS1 protein levels were assessed using Western blotting with an anti-His Ab. Viruses (E) were enumerated using a TCID₅₀ assay, and PRRSV ORF7 (F) was quantified using qRT-PCR. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significance was denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Induction of SOCS1 expression by PRRSV is independent of type I IFNs

Considering that SOCS proteins could be generated by JAK/STAT signaling pathways, which are activated by type I IFN binding to its receptor (56), and previous studies have shown that PRRSV could induce a certain level of IFN-α and IFN-β expression in porcine AMs (57), an investigation was carried out to determine whether PRRSV upregulated SOCS1 expression via IFN signaling pathway. SOCS1 mRNA expression induced by IFN-α and IFN-β was measured using qRT-PCR. As shown in Supplemental Fig. 2A, IFN-α and IFN-β stimulation induced upregulation of SOCS1 at 6, 12, and 24 hpi in porcine AMs. This prompted an analysis to investigate whether SOCS1 transcription might be induced via an auto- or paracrine action of increased type I IFN levels during PRRSV infection.

B18R is a vaccinia virus–encoded soluble type I IFNR that has potent neutralizing activity by acting as a decoy receptor for type I IFNs (49, 50). The cytotoxicity and inhibitory effects of B18R in porcine AMs were first assessed. A cell viability assay indicated that no concentrations of B18R used in this assay caused detectable cell death in porcine AMs (Supplemental Fig. 2B). SOCS1 mRNA expression induced by IFN-α and IFN-β, as well as ISG56 and OAS1a mRNA expression, was nearly abrogated by B18R (Supplemental Fig. 2C–E). Production of SOCS1 protein induced by IFN-α and IFN-β was also significantly decreased by B18R (Supplemental Fig. 2F). These results indicated that B18R can effectively block the signaling pathways downstream of type I IFN, thereby abrogating the expression of SOCS1 induced by type I IFNs. Next, the contribution of PRRSV toward SOCS1 production was examined using B18R. Porcine AMs were preincubated with B18R for 1 h and then inoculated with HN07-1 or poly(I:C), a TLR3 ligand triggering IFN production. At indicated time points, SOCS1 induction was measured by qRT-PCR and ELISA. The upregulation of SOCS1 mRNA and proteins induced by PRRSV in B18R-treated cells was still maintained at high levels. However, the upregulation of SOCS1 mRNA and proteins induced by poly(I:C) was almost completely abolished by B18R (Fig. 3A, 3B). To further confirm this result, siRNA was used to knockdown the expression of IFNAR1 in porcine AMs. The knockdown efficiency of the siRNA, which is specific for silencing the IFNAR1 gene, as assessed by qRT-PCR, was ∼61% (Fig. 3C). Despite partial IFNAR1 silencing, SOCS1 was still upregulated by PRRSV infection in porcine AMs to nearly the same degree, as in cells transfected with an NC siRNA (Fig. 3D, 3E). Similar results were obtained in Marc-145 cells (Fig. 3F, Supplemental Fig. 2G–I). Taken together, these data demonstrated that PRRSV induced SOCS1 production independently of type I IFNs.

FIGURE 3.

Induction of SOCS1 expression by PRRSV is not completely dependent on type I IFNs. (A and B) Porcine AMs were treated or mock treated with B18R for 1 h and then treated with medium alone (control), HN07-1 (MOI = 1), or poly(I:C) at a final concentration of 10 μg/ml. Twenty-four hours later, porcine AMs were harvested and analyzed for SOCS1 mRNA (A) quantification using qRT-PCR and SOCS1 protein (B) quantification using ELISA. (C–E) Porcine AMs were transfected with 20 nM NC siRNA or siRNA against IFNAR1 for 24 h and then infected with PRRSV (MOI = 1). At 24 hpi, porcine AMs were harvested and analyzed for IFNAR1 (C) and SOCS1 mRNA (D) quantification using qRT-PCR or SOCS1 protein (E) quantification using ELISA. (F) Marc-145 cells were pretreated or mock treated with B18R for 1 h and then treated with medium alone (control), HN07-1 (MOI = 1), or poly(I:C) at a final concentration of 10 μg/ml. Twenty-four hours later, the cells were harvested and analyzed for SOCS1 mRNA quantification using qRT-PCR. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Cloning the porcine SOCS1 promoter and mapping potential transcriptional regulatory elements in its 5′-flanking regions

The involvement of IFN in PRRSV-induced SOCS1 production by porcine AMs and Marc-145 cells was ruled out. To further clarify the mechanisms through which SOCS1 transcription was induced by PRRSV, several putative transcriptional regulatory elements were identified in the 5′-flanking regions of the SOCS1 gene (shown in Supplemental Fig. 3A), using Proscan V1.7 and Promoter 2.0. The 5′-flanking regions of porcine SOCS1 extending from nt −2000 to +100 relative to the transcription initiation site were cloned and inserted into a pGL4.17-basic reporter vector to generate the SOCS1 promoter reporter plasmid −2000/100-Luc. Marc-145 cells were transfected with the −2000/100-Luc and pRL-TK Renilla luciferase reporter plasmid. Twenty-four hours later, the transfected Marc-145 cells were harvested and analyzed for luciferase activity. As shown in Supplemental Fig. 3B and 3C, the luciferase activity of the full-length −2000/100-Luc construct exhibited 33.3-fold and 22.3-fold increases over that of pGL4.17-basic in Marc145 cells and CRL2843-CD163, respectively, indicative of the promoter activity of −2000/100-Luc.

To identify the regions of the SOCS1 promoter that were responsible for PRRSV infection, a series of truncated mutants schematically shown in Fig. 4A were constructed. Briefly, −2000/100-Luc contained the full-length promoter region. The −1359/100-Luc excluded one CCAAT-box, one IRF3, one AP-2, and two Sp1 binding sites. The −381/100-Luc contained two IRF3, two Sp1, and one AP-1 binding site. The −58/100-Luc only contained a TATA box on the reverse strand. Following transfection of Marc-145 cells and CRL2843-CD163 cells with these deletion mutants, luciferase activities were evaluated with or without PRRSV infection. The luciferase activities of the −381/100-Luc mutants exhibited 1.8-fold and 2.3-fold upregulation upon PRRSV infection, respectively, whereas other mutants did not (Fig. 4B, 4C). Therefore, the −381/100-Luc construct was used for subsequent studies.

FIGURE 4.

Cloning and sequence analysis of porcine SOCS1 promoter and mapping of potential transcriptional regulatory elements. (A) Schematic representation of the porcine SOCS1 promoter. SOCS1 promoter–truncated mutants were subcloned into pGL4.17-basic vector and the resulting constructs denoted as −2000/100-Luc, −1359/100-Luc, −381/100-Luc, and −58/100-Luc. The relative lengths and positions of the 5′ ends of these fragments were indicated. (B and C) Marc-145 cells (B) or CRL2843-CD163 cells (C) were transfected with a series of SOCS1 promoter–truncated mutants or pGL4.17-basic vector and a pRL-TK Renilla luciferase reporter plasmid. Twenty-four hours later, they were infected or mock infected with HN07-1 (MOI = 1). Another 24 h later, the cells were harvested to determine luciferase activity. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01.

PRRSV N protein induces SOCS1 production

To investigate which PRRSV protein is responsible for SOCS induction, seven HN07-1 nsps or structural protein expression vectors (nsp1α, nsp1β, nsp2, nsp4, nsp5, nsp11, and N) were constructed. These proteins have been reported to be relevant for regulation and evasion of antiviral immune responses (10, 58). Western blotting was performed to assess their expression (Supplemental Fig. 4A, 4B). Marc-145 cells were cotransfected with the plasmid that encodes nsps or structural protein, the luciferase reporter plasmid −381/100-Luc, and the pRL-TK Renilla luciferase reporter plasmid. At 24 h posttransfection, the luciferase activities were assessed. The results show that PRRSV N protein induced the highest SOCS1 promoter activity (Fig. 5A, 5D), which was further confirmed by transfection of Marc-145 cells with an N protein expression vector. Following transfection, SOCS1 protein production was significantly increased in a dose-dependent manner (Fig. 5B).

FIGURE 5.

PRRSV N protein induces SOCS1 production. (A and D) Marc-145 cells (A) or CRL2843-CD163 (D) was cotransfected with a series of plasmids that encode PRRSV nsps and structural protein (nsp1α, nsp1β, nsp2, nsp4, nsp5, nsp11, and N) or pcDNA 3.1-Myc/his_A vector, −381/100-Luc promoter, and pRL-TK Renilla luciferase reporter plasmid. After 24 h, the cells were harvested for luciferase activity analysis. (B) Marc-145 cells were transfected with N protein expression vector at doses of 0.5, 1.0, and 2.0 μg per well (six-well plate) and were harvested at 48 h posttransfection. Western blotting using an anti-SOCS1 Ab was performed to assess SOCS1 protein levels. (C and E) Marc 145 cells (C) or CRL2843-CD163 (E) was cotransfected with PRRSV N protein deletion mutants or pcDNA3.1-Myc/His_A vector, −381/100-Luc promoter, and pRL-TK Renilla luciferase reporter plasmid. After 24 h, the cells were harvested for luciferase activity analysis. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

The PRRSV-2 N protein consists of 123 aa and has two nuclear NLS sequences at positions 10–13 (NLS-1) and 41–47 (NLS-2), N-N at residues 30–37, and an NoLS sequence at positions 41–72 (59, 60). The N protein localizes specifically to the nucleus and nucleolus. To further delineate the essential regions of the N protein involved in SOCS1 production, four deletion mutants of the N protein with mutations in NLS-1, NLS-2, NoLS, and N-N were generated (schematic shown in Supplemental Fig. 4C). The expression of these truncated mutants was confirmed using Western blotting, following transient transfection of Marc145 cells and CRL2843-CD163 cells (Supplemental Fig. 4D, 4E). Marc-145 cells and CRL2843-CD163 cells were cotransfected with each N protein mutant and SOCS1 luciferase reporter plasmid. The abilities of all N protein mutants, except NΔN-N, to activate the −381/100-Luc were significantly attenuated, when compared with the full-length N protein. Moreover, the mutant NΔNLS-2 had no effect on −381/100-Luc activity (Fig. 5C, 5E). This result obtained suggests that the NLS-2 of N protein is essential for SOCS1 promoter activation.

The AP-1 binding motif in the SOCS1 promoter is indispensable for SOCS1 production

Analysis with Proscan V1.7 and Promoter 2.0 revealed that five transcriptional regulatory elements, including two IRF3s binding sites (−308 to −302, −116 to −111), two Sp1 binding sites (−188 to −175, −160 to −151), and one AP-1 binding site (−104 to −96), were found in the −381 to −58 region of the SOCS1 promoter (Fig. 6A). To identify the regulatory elements required for SOCS1 production, a series of mutants defective in each specific transcription regulatory element were prepared (−308ΔIRF3-Luc, −188ΔSp1-Luc, −160ΔSp1-Luc, −116ΔIRF3-Luc, and −104ΔAP-1-Luc). These mutants are shown schematically in Fig. 6A. Marc-145 cells were transfected with the deletion mutants for 24 h. The cells were then inoculated with HN07-1 for 24 h, and luciferase activities were measured. When compared with the wild-type −381/100-Luc, all mutants exhibited low promoter activities (Fig. 6B). When the AP-1 binding site at position −104 to −96 was deleted, the luciferase activity of the SOCS1 promoter (−104ΔAP-1-Luc) induced by PRRSV infection decreased to the same level as the control (pGL4.17-basic), a 55-fold decrease when compared with the intact −381/100-Luc. Similar results were obtained when N protein was used as the stimulant (Fig. 6C). Also, similar results were obtained in CRL2843-CD163 cells (Fig. 6D, 6E). Therefore, these results demonstrated that the AP-1 binding site (−104 to −96) in porcine SOCS1 promoter was indispensable for SOCS1 transcription.

FIGURE 6.

AP-1 binding element in the porcine SOCS1 promoter is indispensable for SOCS1 production. (A) Schematic diagram representing porcine SOCS1 promoter deletion mutants (−308ΔIRF3-Luc, −188ΔSp1-Luc, −160ΔSp1-Luc, −116ΔIRF3-Luc, −104ΔAP-1-Luc, and −381/100-Luc). (B and D) Marc-145 cells (B) or CRL2843-CD163 cells (D) were cotransfected with a series of SOCS1 promoter mutants and pRL-TK Renilla luciferase reporter plasmid for 24 h and then infected or mock infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for luciferase activity analysis. (C and E) Marc-145 cells (C) or CRL2843-CD163 cells (E) were cotransfected with a series of SOCS1 promoter mutants, N protein, and pRL-TK Renilla luciferase reporter plasmid. Twenty-four hours later, the cells were harvested for luciferase activity analysis. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

AP-1 is critical for PRRSV and N-induced SOCS1 expression

Because the AP-1 binding site in porcine SOCS1 was important for PRRSV-induced SOCS1 promoter activity, it was hypothesized that AP-1, the dimeric complex of c-Jun and c-Fos, might be involved in porcine SOCS1 induction during PRRSV infection. To test this hypothesis, porcine AMs were pretreated with a specific c-Jun inhibitor (SR11302) for 1 h prior to HN07-1 infection. Twenty-four hours later, SOCS1 mRNA expression was analyzed using qRT-PCR. As shown in Fig. 7A, SR11302 significantly inhibited SOCS1 induction by PRRSV in a dose-dependent manner. The cytotoxicity of the inhibitor was determined by a cell viability assay. No concentrations of the inhibitor caused detectable cell death in porcine AMs (Supplemental Fig. 4F) and Marc-145 cells (data not shown). The roles of c-Jun and c-Fos in PRRSV-induced SOCS1 production were confirmed by evaluating SOCS1 expression following the knockdown of c-Jun and c-Fos expression, using siRNA prior to PRRSV infection. The knockdown efficiencies of siRNAs were assessed using qRT-PCR and Western blotting (Fig. 7B, 7C). Knockdown of c-Jun and c-Fos significantly dampened SOCS1 mRNA and protein upregulation in porcine AMs (Fig. 7D, 7E). AP-1 inhibitor SR11302 also significantly decreased N protein–induced SOCS1 production in Marc-145 cells (Fig. 7F). Next, an investigation was carried out to determine whether AP-1 pathway was activated by PRRSV or the N protein. Porcine AMs were infected with HN07-1 and then harvested at 12 and 24 hpi. The phosphorylations of c-Jun and c-Fos were assessed using Western blotting. As expected, c-Jun and c-Fos phosphorylations increased (Fig. 7G), as previously reported (61). A similar extent of c-Jun phosphorylation was observed when Marc-145 cells were transfected with an expression vector that encodes the PRRSV N protein (Fig. 7H). The level of phosphorylation of c-Fos in Marc-145 cells was difficult to detect. Taken together, these results indicate that both PRRSV and the N protein activate the AP-1 pathways, which was indispensable for SOCS1 production.

FIGURE 7.

The AP-1 signaling pathway is involved in SOCS1 upregulation by PRRSV. (A) Porcine AMs were pretreated with DMSO or SR11302 for 1 h and then infected or mock infected with HN07-1 (MOI = 1) in the presence of inhibitor. The cells were harvested at 24 hpi for analysis of SOCS1 mRNA levels using qRT-PCR. (B–E) Porcine AMs were transfected with 20 nM NC siRNA or siRNAs against porcine c-Jun and c-Fos using RNAiMAX and then infected after 24 h with PRRSV HN07-1 (MOI = 1). After 24 h, the cells were harvested and analyzed to measure knockdown efficiency (B and C). Or the cells were harvested for analysis of SOCS1 mRNA levels (D) using qRT-PCR or lysed for SOCS1 protein (E) quantification using ELISA. (F) Marc-145 cells were pretreated with DMSO or SR11302 for 1 h and transfected with a vector that encodes the PRRSV N protein. After 24 h, the cells were harvested for SOCS1 protein level and p–c-Jun/c-Jun and p–c-Fos/c-Fos level quantification using Western blotting. (G) Porcine AMs were infected with HN07-1 (MOI = 1) and then harvested at the indicated time points for analysis of SOCS1, p–c-Jun/c-Jun, and p–c-Fos/c-Fos levels, using Western blotting. (H) Marc-145 cells were transfected with a vector that encodes the PRRSV N protein at doses of 0.5, 1.0, and 2.0 μg per well (six-well pates) and then harvested 48 h later for analysis of SOCS1 and p–c-Jun/c-Jun levels using Western blotting. Relative expression levels shown below the images were evaluated as fold changes after normalization to β-actin or GAPDH levels. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

p38/AP-1 and JNK/AP-1 signaling pathways are involved in PRRSV-induced SOCS1 expression

The upstream signaling pathways involved in PRRSV-induced SOCS1 expression were investigated. MAPK signaling pathways mainly include the ERK, p38, and JNK signaling pathways. All three signal transduction pathways have been reported to be activated during PRRSV infection (61–65). Inhibitors were used to specifically inhibit each MAPK signaling pathway. The cytotoxicity of signaling pathway inhibitors toward porcine AMs was investigated. No concentrations of the inhibitors used in this study caused detectable cell death (Supplemental Fig. 4G). Porcine AMs were pretreated with the inhibitors of ERK, p38, and JNK for 1 h prior to PRRSV infection. Twenty-four hours later, SOCS1 mRNA and protein levels were assessed using qRT-PCR and ELISA, respectively. The p38 inhibitor SB203580 and the JNK inhibitor SP600125 significantly inhibited SOCS1 expression in porcine AMs, whereas the ERK inhibitor PD98059 had no effect on PRRSV-induced SOCS1 expression (Fig. 8A, 8B). These data demonstrated that p38 and JNK MAPK signaling pathways were involved in SOCS induction by PRRSV.

FIGURE 8.

The p38 and JNK MAPK signaling pathways are required for PRRSV upregulation of SOCS1 expression. (A and B) Porcine AMs were pretreated with DMSO, p38 inhibitor SB203580 (SB), ERK inhibitor PD98059 (PD), or JNK inhibitor SP600125 (SP) for 1 h and then infected or mock infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for analysis of SOCS1 mRNA levels (A) using qRT-PCR and SOCS1 protein levels (B) using ELISA. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by **p < 0.01.

The relationships between p38, JNK, and AP-1 during PRRSV infection were investigated. Porcine AMs were pretreated with different doses of the p38 inhibitor SB203580 or JNK inhibitor SP600125 for 1 h before PRRSV infection. At 24 hpi, c-Jun, c-Fos, phospho-c-Jun, and phospho-c-Fos levels were analyzed using Western blotting. The results obtained show that JNK inhibitor (SP600125) but not p38 inhibitor (SB203580) significantly impaired PRRSV-induced c-Jun phosphorylation in a dose-dependent manner (Fig. 9A, 9D). Moreover, p38 inhibitor (SB203580) but not JNK inhibitor (SP600125) significantly impaired PRRSV-induced c-Fos and c-Fos phosphorylation in a dose-dependent manner (Fig. 9B, 9E). Both p38 inhibitor (SB203580) and JNK inhibitor (SP600125) dose-dependently inhibited PRRSV-induced SOCS1 production (Fig. 9C, 9F). These data indicated that c-Jun activation by PRRSV is mediated through the JNK pathway and that c-Fos activation is mediated through the p38 pathway.

FIGURE 9.

The p38/AP-1 and JNK/AP-1 signaling pathways are involved in PRRSV-induced SOCS1 production. (A–C) Porcine AMs were pretreated with DMSO or p38 inhibitor SB203580 (SB) for 1 h and then infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for analysis of p–c-Jun/c-Jun (A), p–c-Fos/c-Fos (B), and SOCS1 levels (C) using Western blotting. (D–F) Porcine AMs were pretreated with DMSO or JNK inhibitor SP600125 (SP) for 1 h and then infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for analysis of p–c-Jun/c-Jun (D), p–c-Fos/c-Fos (E), and SOCS1 levels (F) using Western blotting. (G and H) Marc-145 cells were pretreated with DMSO, p38 inhibitor SB, or JNK inhibitor SP for 1 h, and they were then transfected with plasmid that encodes the PRRSV N protein at a dose of 2.0 μg per well (six-well plates) for 48 h. The cells were harvested for analysis of SOCS1 and p–c-Jun/c-Jun expression using Western blotting. Relative expression levels shown below the images were evaluated as fold changes after normalization to GAPDH levels.

An investigation was also carried out to determine whether p38 and JNK were involved in N protein–induced SOCS1 expression. Marc-145 cells were pretreated with p38 inhibitor (SB203580) or JNK inhibitor (SP600125) for 1 h prior to transfection with PRRSV N protein plasmid. At 48 h posttransfection, the cells were collected for analysis of c-Jun, p–c-Jun, and SOCS1 levels using Western blotting. As shown in Fig. 9G and 9H, c-Jun phosphorylation induced by N protein was significantly impaired by JNK inhibitor (SP600125) but not p38 inhibitor (SB203580) in a dose-dependent manner, suggesting that activation of c-Jun by PRRSV N protein was mediated by the JNK pathway. Meanwhile, PRRSV N protein–induced SOCS1 expression was also significantly impaired by SP600125 (Fig. 9H).

In summary, these results indicate that PRRSV infection induces SOCS1 production through the p38/AP-1 and JNK/AP-1 signaling pathways. The PRRSV N protein induces SOCS1 production through the JNK/AP-1 signaling pathway.

Discussion

To evade innate immune responses, viruses hijack cellular proteins to impair host immunity to establish persistent infection. In this study, it has been shown that PRRSV promoted self-replication through a mechanism involving SOCS1 induction. It was observed that PRRSV infection strongly induced SOCS1 production in a time- and dose-dependent manner (Fig. 1). SOCS1 upregulation by PRRSV was not fully dependent on type I IFNs but was involved in p38/AP-1 and JNK/AP-1 signaling pathways (Figs. 7–9). Using luciferase reporter assays, the thought that PRRSV N protein might significantly upregulate SOCS1 expression was completely screened out (Fig. 5, Supplemental Fig. 4A–E). It was further proven that NLS-2 of N protein played a critical role in SOCS1 promoter activation (Fig. 5). Taken together, the results of this study uncovered an unappreciated mechanism employed by PRRSV to suppress host immune response and enable efficient virus replication.

Because PRRSV has a very narrow cellular tropism, with a preference for monocyte/macrophage lineage cells and particularly for porcine AMs in the lung (66), the ability of PRRSV (including PRRSV-1 and PRRSV-2 species) to induce SOCS1 production in porcine AMs was first assessed. It was observed that all tested representative PRRSV-1 and PRRSV-2 strains (GZ11-G1, HN07-1, BJ-4, and HNhx) significantly upregulated SOCS1 production. However, the increase of SOCS1 in Marc-145 cells and CRL2843-CD163 cells during PRRSV infection showed delayed effects (36 hpi), when compared with those in porcine AMs. The increase of SOCS1 in porcine AMs upon PRRSV-1 strain GZ11-G1 infection also showed a delayed effect (after 24 hpi), when compared with PRRSV-2 strain infection. These data suggest differences in SOCS1 expression among different cells or PRRSV strains. Moreover, SOCS1 expression during the early stages of PRRSV infection (3 hpi on porcine AMs or 6 and 12 hpi on Marc-145 cells) exhibited slightly decreased effects (Fig. 1A, 1E). It was speculated that during early infection stage, when the first round of PRRSV replication remained incomplete, SOCS1 downregulation is a beneficial host response to PRRSV infection (67). Indeed, SOCS1 upregulation requires PRRSV replication (Fig. 1I). SOCS1 functions as a crucial intracellular protein that negatively modulates innate and adaptive immunity. Most previous studies have demonstrated that SOCS1 mainly targets the JAK-STAT signaling pathways. Recent studies have presented direct evidence that SOCS1 downregulates type I IFN production (24). The results of this study indicate that SOCS1 not only suppressed IFN-β expression but also inhibited the expression of ISGs, which are stimulated by IFN (Supplemental Fig. 1). Several other proteins that act as negative regulators of immune response have been reported to be modulated by PRRSV infection. For example, IL-10, which is known to negatively regulate the production of numerous inflammatory cytokines, cell-mediated immune responses, and Ag presentation, was upregulated in PRRSV-infected porcine AMs (68, 69). Secretion of soluble CD83, which is involved in immunosuppression of T cell proliferation and differentiation, was strongly stimulated by PRRSV and PRRSV proteins (N and nsp10) (70, 71). Thus, it was speculated that all these factors caused a comprehensive and complex immunosuppressive environment in vivo during PRRSV infection, which together contribute to PRRSV replication and establishment of persistent infection. However, the detailed mechanisms underlying the inhibitory effects of SOCS1 on IFN and whether PRRSV-induced SOCS1 upregulation acts on other signaling pathways need to be further elucidated. Indeed, PRRSV propagation was strongly enhanced by SOCS1 (Fig. 2). The results obtained in this study add a new dimension to scientific knowledge of the strategies used by PRRSV to antagonize type I IFN responses.

The multifunctional PRRSV N protein is the most abundant viral protein in infected cells and is highly immunogenic in pigs (72). The N protein was reported to be involved in induction of IL-10, IL-15, and regulatory T lymphocyte differentiation (71, 73). Moreover, the N protein suppressed IFN-β expression through inhibition of IRF3 phosphorylation and nuclear translocation (74). In this study, the results obtained show that PRRSV N protein could significantly activate SOCS1 promoter activity and induce SOCS1 expression at the protein level in Marc-145 cells (Fig. 5). These results demonstrate that N protein also plays an important role in SOCS1 expression, reflecting a new function of this protein. However, viral 5′ triphosphate RNA is reported to be the major transcriptional inducer of another SOCS family protein (SOCS3) and causes reduced STAT1 phosphorylation during influenza A virus infection (39). Further studies on the ability of viral RNAs to induce SOCS1 expression are needed.

In the presence of B18R (which inhibits the activities of type I IFNs) or siRNAs (which silence IFNAR1 expression), the PRRSV still significantly induced SOCS1 upregulation in porcine AMs and Marc-145 cells (Fig. 3). Because B18R almost completely inhibited IFN-induced ISG expression and SOCS1 production, it was concluded that SOCS1 induction by PRRSV is hardly dependent on type I IFNs. The SOCS1 induction by PRRSV is a primary response, not a secondary effect induced by the secretion of IFN. However, the possibility that the induction of SOCS1 by PRRSV might minimally or partly depend on IFN signaling pathways cannot be ruled out. The results obtained in this study indicated that p38/AP-1 and JNK/AP-1 signaling pathways were involved in PRRSV-induced SOCS1 production. JNK/AP-1 signaling pathway was involved in N protein–induced SOCS1 production. Because the signaling pathways used for SOCS1 induction by PRRSV and N protein differed, it was speculated that other factors might have participated in the SOCS1 induction. Therefore, there is need for further investigation.

In conclusion, to our knowledge, it has been reported in this article for the first time that PRRSV and PRRSV N protein are able to induce SOCS1 production, which is exploited by PRRSV to facilitate the replication of PRRSV through a molecular mechanism that involves AP-1 signaling pathway but not the autoregulatory action of type I IFNs. The findings of this study provide novel (to our knowledge) insights into the strategies employed by PRRSV to subvert the host immune response.

Disclosures

The authors have no financial conflicts of interest.