Suppression of immune responses in pigs by nonstructural protein 1 of Porcine reproductive and respiratory syndrome virus

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is characterized by a delayed and defective adaptive immune response. The viral nonstructural protein 1 (NSP1) of the PRRS virus (PRRSV) is able to suppress the type I interferon (IFN) response in vitro. In this study, recombinant adenoviruses (rAds) expressing NSP1 (rAd-NSP1), glycoprotein 5 (GP5) (rAd-GP5), and the NSP1-GP5 fusion protein (rAd-NSP1-GP5) were constructed, and the effect of NSP1 on immune responses was investigated in pigs. Pigs inoculated with rAd-NSP1 or rAd-NSP1-GP5 had significantly lower levels of IFN-γ and higher levels of the immunosuppressive cytokine IL-10 than pigs inoculated with rAd-GP5, wild-type adenovirus, or cell culture medium alone. The antibody response to vaccination against classic swine fever virus (CSFV) was significantly decreased by inoculation of NSP1 7 d after CSFV vaccination in pigs. Thus, NSP1-mediated immune suppression may play an important role in PRRSV pathogenesis.

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) is a small, enveloped, single-stranded, positive-sense RNA virus (1,2) in the genus Arterivirus of the family Arteriviridae (3). It causes economically important disease in pigs that is characterized by a delayed and defective adaptive immune response (4,5). A highly pathogenic PRRSV, which first emerged in China, has caused heavy economic losses in many pig-producing regions (6,7).

The PRRSV genome is approximately 15 kb long and contains 9 open reading frames (ORFs) flanked by untranslated regions at the 5′ and 3′ termini (8–10); ORF1a and ORF1b, situated at the 5′ end, constitute nearly 80% of the viral genome and encode viral nonstructural proteins (NSPs) involved in viral polyprotein processing and replication (11–13). The complete processing of the polyproteins is predicted to yield 12 NSP polypeptides, NSP1 to NSP12 (14–17). Among the polypeptides, NSP1 is critical for subgenomic mRNA synthesis (3). It contains papain-like proteinase α (PCPα), which directs the release of NSP1α (20 kDa), along with PCPβ, which directs the release of NSP1β (27 kDa), depending on the activities of PCPα, and a zinc-finger motif required for subgenomic mRNA transcription (18).

Because type I interferon γ (IFN-γ) is a signature cytokine of the T helper cell Th1-associated response, it is a useful indicator of cell-mediated immunity (CMI) (19). The immunosuppressive cytokine IL-10 can suppress IFN-γ production in peripheral blood mononuclear cells (PBMCs) in pigs (20). The production of IL-10 has been reported to increase after PRRSV infection, the increase correlating with reduced IFN-γ production in virus-infected cells (21). In addition, PRRSV infection can suppress the antibody response to vaccination against classic swine fever virus (CSFV), the most common means of preventing and controlling this important disease of domestic pigs in epidemic areas (22,23), and result in vaccination failure when the pigs are subsequently exposed to CSFV (24,25).

Since NSP1 is expressed early in the virus life cycle, it is available to the macrophage proteosome machinery from the earliest time of infection for degradation and presentation to the immune system in the context of major histocompatibility classes I and II (26,27). This polypeptide is critical to the virus’s life cycle and likely to be toxic to cells owing to its protease activities. It can be processed as NSP1α and NSP1β, and NSP1β is the main protein antagonizing cellular production of type I IFN (28,29). The aim of this study was to determine if PRRSV NSP1 expressed in an adenovirus is able to suppress humoral and CMI responses in pigs.

Materials and methods

Cell cultures and viruses

Recombinant and wild-type adenoviruses (rAd and wtAd) were grown in human embryo kidney (HEK-293A) cells. Highly pathogenic PRRSV strain SY0608 was grown in MARC-145 cells. This strain, belonging to type 2, was first isolated in mideastern China. It caused illness and death in 100% and 25% to 50%, respectively, of pigs 30, 65, and 105 d old, as well as the birth of stillborn and weak piglets. The NSP2 contained 2 discontinuous deletions, 1 and 29 amino acids long, corresponding to strain VR-2332, positions 480 and 531 to 559, respectively (6). Dulbecco’s modified Eagle’s essential medium with 10% heat-inactivated fetal calf serum (FCS) was added to the cell cultures, which were then incubated at 37°C in 5% CO₂. Cell lines were inoculated 24 h after seeding.

Amplification and cloning of the PRRSV NSP1 and glycoprotein 5 (GP5) genes

Viral RNA was extracted with the use of TRIzol (Invitrogen, Carlsbad, California, USA). Reverse transcription (RT) was performed at 50°C for 60 min, and 20 μL of the RT mixture was added, for a final concentration of 2 μg of total RNA, along with 200 U of SuperScript III RT (Gibco BRL, Burlington, Ontario), 2.5 μM of oligo(dT)12-18, 5 mM of dithiothreitol, 1× RT buffer, and 0.5 mM of each deoxynucleotide triphosphate (dNTP).

Sequences of primer pairs for amplification of the PRRSV SY0608 NSP1 and GP5 genes were as follows: NSP1, forward 5′-GAGAGATCTATGTCTGGGATACTTG-3′ (BglII site underlined) and reverse 5′-TATAAGCTTACCGTACCACTTATGACTG-3′ (HindIII site underlined); GP5-1, forward 5′-GAAAGATCTATGTT GGGGAAGTGCT-3′ (BglII site underlined) and reverse 5′-GAGAAGCTTGAGACGACCCCATTG-3′ (HindIII site underlined); and GP5, forward 5′-GAAAAGCTTATGTTGGGGAAGTGC-3′ (HindIII site underlined) and reverse 5′-GAGGATATCGAGA CGACCCCATTG-3′ (EcoRV site underlined) (GenBank EU144079).

Amplification was performed in a 50-μL reaction mixture containing 1.5 mM of MgCl₂, 1× polymerase chain reaction (PCR) buffer, 0.2 mM of each dNTP, 20 pM of each primer, 1.5 U of Taq DNA polymerase (Promega, Madison, Wisconsin, USA), and 2 μL of complementary DNA. A PTC-150 thermocycler (MJ Research, South San Francisco, California, USA) was used with the following program: denaturation at 94°C for 5 min, 30 cycles composed of denaturation at 94°C for 40 s, annealing at 60°C for 40 s, and extension at 72°C for 1 min, and a final extension at 72°C for 10 min.

The PCR products were cloned into the vector pShuttle-CMV, which produced 3 recombinant plasmids, pShuttle-CMV-NSP1, pShuttle-CMV-GP5, and pShuttle-CMV-NSP1-GP5. The recombinant plasmids were sequenced to confirm tandem in-frame insertion of the NSP1 and GP5 genes.

Construction of rAd expressing NSP1 and/or GP5

Recombinant adenoviruses were generated as described previously (30,31), with some modifications. The recombinant shuttle vector pShuttle-CMV-NSP1 was linearized with PmeI and cotransformed with the adenovirus backbone plasmid pAdEasy-1 (Stratagene, La Jolla, California, USA) into Escherichia coli BJ5183 by electroporation. The rAd vector was generated by homologous recombination, and the positive clones were identified by plasmid extraction and enzyme digestion with PacI. The resulting adenoviral plasmid was linearized with PacI and transfected into HEK-293A cells in 24-well plates with the use of TransFast Transfection Reagent (Promega). The yield of adenovirus was observed under the microscope, and the 50% tissue culture infectious dose (TCID₅₀) was determined in HEK-293A cells by the Reed–Muench method.

Identification of NSP1 and GP5 expressed in the constructs

Indirect immunofluorescence assay (IFA)

Expression of NSP1 and GP5 in HEK-293A cells infected with rAd was determined as described previously (31). The monoclonal antibody against PRRSV NSP1 was produced in our laboratory and had high specificity for PRRSV, as determined by IFA (1:200 dilution). The polyclonal antibody against PRRSV GP5 was produced in our laboratory by vaccination of mice with purified truncated GP5 expressed by pGEX-6P-1 in E. coli BL21 [1:200 diluted in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T)].

The HEK-293A cells, seeded in 96-well plates, were fixed in ethanol. After 3 washes in PBS (pH 7.4) the fixed cells were incubated with antibodies against NSP1 and GP5 for 1 h at 37°C. Unbound antibodies were washed 3 times with PBS-T. Fluorescein-conjugated goat antibody against mouse antigen (Boston Bio-Tech, Boston, Massachusetts, USA) was added and the mixture incubated for 1 h at 37°C. After 3 washes with PBS, positive signals were sought by fluorescence microscopy with a Zeiss Axiovert 200 (Carl Zeiss Microscopy, Göttingen, Germany).

Western blot analysis

Western blot analysis was performed as described previously, with some modifications (32). Lysates of HEK-293A cells infected with rAd or wtAd were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Pall Life Sciences, Ann Arbor, Michigan, USA). The membranes were incubated with PRRSV-specific antiserum (porcine anti-PRRSV, produced in our laboratory; 1:100 diluted in PBS-T) at 37°C for 2 h and then at 37°C for 1 h with horseradish peroxidase-conjugated staphylococcal protein A (Boshide, Wuhan, China). Positive signals were detected with the use of luminal-based chemiluminescence reagents (SuperSignal West Pico Trial Kit; Thermo Fisher Scientific Pierce Protein Research Products, Rockford, Illinois, USA).

Effect of PRRSV NSP1 on CMI and humoral immunity

All experimental protocols were approved by the Institutional Animal Care and Ethics Committee of Nanjing Agricultural University (permit IACECNAU20090905) and met the International Guiding Principles for Biomedical Research Involving Animals (33).

Cell-mediated immunity

Twenty-five 3-week-old piglets free of PRRSV, Porcine circovirus-2, and adenovirus infections (serum antibody tests and PCR for these organisms all gave negative results) were divided into 5 equal groups and housed in separate rooms. Three groups were inoculated with 10⁷ TCID₅₀ of rAd-NSP1, rAd-GP5, or rAd-NSP1-GP5 and given a booster 14 d later. The other 2 groups were inoculated with either wtAd or the cell culture medium (mock control) with the same protocol.

On days 0 (day of inoculation), 7, 14, 21, and 28, PBMCs were isolated from 5 mL of blood collected from the pigs into heparin-containing tubes. Viable cells were counted by the trypan blue exclusion method. The cells were resuspended in RPMI 1640 (Gibco BRL) supplemented with 10% FCS, 2 mM of L-glutamine, 50 mM of 2-mercaptoethanol, 100 U/mL of penicillin, and 100 mg/mL of streptomycin solution (Gibco BRL) and seeded in triplicate in 96-well flat-bottom plates, 100 mL per well. After stimulation with concanavalin A (5 μg/mL) for 24 h, supernatant was collected and the concentrations of IFN-γ and IL-10 were determined quantitatively with the use of commercially available porcine cytokine enzyme-linked immunosorbent assay (ELISA) kits (Invitrogen).

Humoral immune response

Thirty 3-week-old piglets free of PRRSV, adenovirus, and CSFV infections were divided into 6 equal groups and housed in separate rooms. Four groups were inoculated intramuscularly with 2 mL of a modified live lapinized Chinese-strain CSFV vaccine and then with 10⁷ TCID₅₀ of rAd-NSP1, rAd- GP5, rAd-NSP1-GP5, or wtAd 7 d later. The other 2 groups were inoculated with either CSFV vaccine only or the cell culture medium only (mock control) with the same inoculation protocol. At 14, 21, and 28 d after the 1st inoculation, blood samples were collected and the levels of antibody to CSFV determined in the serum with a commercially available blocking ELISA kit (HerdChek CSFV Ab; IDEXX Laboratories, Westbrook, Maine, USA). Samples were considered to be positive at a calculated blocking percentage ≥ 40% and negative at a blocking percentage ≤ 30%.

Statistical analysis

The differences in the level of humoral responses and cytokine production between the groups were determined by 1-way repeated-measures analysis of variance and Duncan’s test as a post-test with SPSS software, version 17.0 (SPSS, Chicago, Illinois, USA). Differences were considered to be significant at P < 0.05.

Results

The titers of rAd-NSP1 (expressing NSP1), rAd-GP5 (expressing GP5), and rAd-NSP1-GP5 (expressing NSP1-GP5) were 10^9.0, 10^8.0, and 10^9.0 TCID₅₀/mL, respectively. Expression of NSP1 was confirmed by IFA (Figure 1) and Western blot analysis (Figure 2). The PRRSV NSP1 was cleaved into α and β subunits of approximately 20 and 27 kDa, respectively.

Figure 1.

Results of indirect immunofluorescence assay of monolayers of human embryo kidney (HEK-293A) cells infected with recombinant adenoviruses (rAds) expressing nonstructural protein 1 (NSP1) of the Porcine reproductive and respiratory syndrome virus (PRRSV) (rAd-NSP1), PRRSV glycoprotein 5 (GP5) (rAd-GP5), and the NSP1-GP5 fusion protein (rAd-NSP1-GP5). The assays were done with NSP1-specific monoclonal antibody (left panels) and mouse anti-GP5 serum (right panels). Noninfected HEK-293A cells and cells infected with wild-type adenovirus (wtAd) were used as controls.

Figure 2.

Western blot analysis of cell lysates infected with the same rAds and incubated with PRRSV-specific porcine antiserum. Lane 1 — cell lysates of rAd-NSP1-GP5; lane 2 — cell lysates of rAd-GP5; lane 3 — cell lysates of rAd-NSP1; lane 4 — cell lysates of wtAd. Arrows indicate the expected molecular sizes of NSP1β-GP5, GP5, NSP1β, and NSP1α. Proteins standards are shown on the left.

At 21 and 28 d after inoculation the mean concentration of IFN-γ produced by PBMCs was significantly lower and the mean concentration of IL-10 significantly higher in the pigs inoculated with rAd- NSP1 and rAd-NSP1-GP5 than in those inoculated with rAd-GP5, wtAd, or cell culture medium (P < 0.05) (Figure 3).

Figure 3.

Concentrations of interferon (IFN)-γ and interleukin (IL)-10 in the supernatants of peripheral blood mononuclear cells (PBMCs) isolated from pigs at various times after inoculation with the rAds as well as cell culture medium (“Mock”) and wtAd as controls. After stimulation with concanavalin A in triplicate, enzyme-linked immunosorbent assays (ELISAs) were done with the use of commercial kits. Data are shown as means ± standard errors froms 3 independent experiments. Arrows indicate time of initial and booster inoculations. Different letters indicate a significant difference at P < 0.05.

Anti-CSFV antibodies were detected in serum from the pigs inoculated with CSFV vaccine alone or the cell culture medium alone, as well as in those that were inoculated with rAd-NSP1, rAd-GP5, or rAd-NSP1-GP5 7 d after CSFV vaccination. The antibody levels increased rapidly in the CSFV-vaccinated groups in the 2 wk after vaccination (Figure 4). The levels were significantly lower (P < 0.05) in the CSFV + rAd-NSP1 and CSFV + rAd-NSP1-GP5 groups than in the CSFV alone and CSFV + rAd-GP5 groups during the period 2 to 3 wk after vaccination.

Figure 4.

Levels of serum antibodies, measured with a commercial ELISA kit, against classic swine fever virus (CSFV) after CSFV vaccination and after inoculation 7 d later with the rAds or with wtAd. Mock vaccination with cell culture medium was used as a control procedure. Data are shown as means ± standard errors from 3 independent experiments. The dashed line indicates the cut-off blocking percentage (40%). Different letters indicate a significant difference at P < 0.05.

Discussion

Previous studies have shown that PRRSV infection elicits a poor innate response of antiviral type I IFN, which was postulated to result in a weak adaptive immune response, as demonstrated by a short duration of CMI responses (34,35) and slow development of a virus-specific IFN-γ response (36). In vitro, PRRSV infection of porcine alveolar macrophages (PAMs), monocyte-derived macrophages, and MARC-145 cells inhibited IFN-α and IFN-β production (5,18,21). In transfection experiments using recombinant plasmids expressing the 10 individual PRRSV NSPs, Beura et al (29) showed that 4 PRRSV NSPs (NSP1, NSP2, NSP4, and NSP11) contribute to IFN antagonism. However, there is little information on the immune response to PRRSV NSPs in vivo. In this study, rAds expressing NSP1, the amino terminal protein in a polyprotein encoded by PRRSV, that were derived from highly pathogenic PRRSV were constructed and immune responses determined in pigs. The animals inoculated with rAd-NSP1 and rAd-NSP1-GP5 had significantly lower levels of IFN-γ than those inoculated with rAd-GP5, wtAd, or cell culture medium alone. Pigs inoculated with rAd-NSP1 and rAd-NSP1-GP5 had greater secretion of IL-10 than those inoculated with rAd-GP5, wtAd, or medium alone.

The self-cleavage products of NSP1 during virus infection, NSP1α and NSP1β, could moderate inhibitory effects on IFN-β promoter activation. When expressed stably in cell lines, they strongly inhibited double-stranded RNA signalling pathways (28). Moreover, NSP1β is the main protein antagonizing cellular production of type I interferon, and it inhibits both IFN regulatory factor 3 and NF-κB-dependent gene induction by double-stranded RNA and Sendai virus (29). Beura et al (29) proposed that NSP1β modulates the host innate immune response by antagonizing IRF3 activation. In this study we found that NSP1, which plays a key role in CMI responses (37), could significantly inhibit the secretion of IFN-γ in pigs inoculated with rAd-NSP1 or rAd-NSP1-GP5 compared with pigs inoculated with rAd-GP5 or wtAd. To clarify the role of NSP1α and NSP1β in CMI responses in pigs in vivo, these proteins should be expressed individually with the adenovirus and their effects determined. In addition, to confirm the innate immunity of NSP1 found in vitro (29), other innate cytokines, such as IFN-1β and IL1, should be assayed in pigs.

The immunomodulatory cytokine IL-10 inhibits the synthesis and release of other cytokines (38). Increased production of IL-10 induced by PRRSV infection might be one of the strategies used by the virus to modulate the host’s immune responses, thereby contributing to the unique clinical picture observed after PRRSV infection (39). This cytokine contributed to significantly reduced IFN-γ and TNF-α expression by T lymphocytes (40,41). In our study, NSP1 significantly enhanced secretion of IL-10 in pigs inoculated with rAd-NSP1 and rAd-NSP1-GP5 compared with those inoculated with rAd-GP5 and wtAd. Therefore, the high expression of IL-10 observed in the present study may be responsible for the reduced expression of IFN-γ, which in turn may prolong viral replication in pigs.

The antibody response to CSFV plays an important role in protective immunity (22). To understand the effect of NSP1 on the humoral immune responses, 3-wk-old piglets free of PRRSV, CSFV, and adenovirus infections were inoculated with rAds 7 d after CSFV vaccination and the CSFV-specific antibodies assayed. The levels of antibodies to CSFV were negatively affected by inoculation with rAd-NSP1 and rAd-NSP1-GP5. This finding may be relevant to vaccination programs for the prevention and control of CSF and PRRS.

Previously it was shown that GP5 is the most important glycosylation protein of PRRSV involved in the generation of humoral and CMI responses (42) and that fusion expression of PRRSV GP3 and GP5 delivered by a human adenovirus vector enhances immune responses in pigs (43). Usually neutralizing antibodies play an important role in immune protection. Although GP5 proteins have neutralizing epitopes, only weak and delayed neutralizing antibodies could be induced by constructs expressing GP5 alone (44,45). In this study, GP5 was used as a positive control to examine the effect of NSP1 on adaptive immunity. Neutralizing antibodies to PRRSV were not detected during the period of observation (data not shown).

Conclusion

The PRRSV NSP1 suppressed IFN-γ secretion and increased IL-10 secretion in pigs. It also inhibited the antibody response to CSFV when inoculated 7 d after CSFV vaccination. Thus, NSP1-mediated immune suppression may play an important role in the pathogenesis of PRRS.