Porcine reproductive and respiratory syndrome virus (PRRSV), a positive-sense single-stranded, enveloped RNA virus that is a member of the family Arteriviridae
), causes the most economically significant infectious malady afflicting pigs in commercial swine farms worldwide (2
). Exposure of the respiratory mucosa of a pig to PRRSV results in virus replication in regional macrophages (Mϕ) and the development of viremia within 12 h after infection, leading to systemic distribution of the virus to other macrophage populations in the body (3
). In the lung, PRRSV exploits alveolar macrophages (AMϕ) for its replication, triggering a massive infiltration of the alveolar septa by macrophages, resulting in interstitial pneumonia (5
). In the absence of secondary bacterial infections, pneumonias caused by PRRSV are rarely lethal and begin to resolve within 2 weeks (6
). While interleukin 1 (IL-1) and IL-6 are amply detected in bronchoalveolar lavage (BAL) fluids obtained from such pneumonic lungs, the presence of alpha interferon (IFN-α) and tumor necrosis factor alpha (TNF-α) is negligible (8–12
). In contrast, pneumonias caused by PRRSV that are accompanied by a secondary bacterial infection result in a severe respiratory syndrome characterized by abundant presence of TNF-α in the lung, enhanced lung tissue damage, high morbidity, hypoxia, and a high rate of mortality (6
). The mechanism responsible for the apparent pathogenic synergy between PRRSV and bacterial pathogens is not understood (15
Compared to the profile of innate cytokines elicited by other viruses that cause pneumonia in pigs, such as swine influenza virus and porcine respiratory coronavirus, which trigger the abundant presence of IFN-α and TNF-α in lung tissue (5
), the nominal presence of these two cytokines in the lungs of pigs afflicted by PRRSV is intriguing; however, the mechanism responsible for this condition is unclear (16
). Given the critical roles that IFN-α and TNF-α play in host immunity, the apparent ability of PRRSV to modulate the production of the two cytokines has been extensively examined. Several studies have relied on measuring transcription factor (TF) activation using reporter gene assays and overexpression of single viral genes. These studies indicate that some PRRSV nonstructural proteins have the ability to modulate cytokine production stimulated by strong agonists, like synthetic double-stranded RNA (dsRNA) or lipopolysaccharide (LPS), by inhibiting the activation of IRF3 or NF-κB (17–20
). In the context of virus infection, the modulatory properties ascribed to PRRSV have been found to be disparate. For example, in the case of IFN-α, virus infection has been reported to inhibit the production of the cytokine in response to stimulation with potent type I IFN agonists, such as porcine coronavirus (8
) and synthetic dsRNA (21
). On the other hand, the production of TNF-α in response to stimulation with LPS has been reported to range from enhancement to inhibition (22
). To clarify these disparate modulatory outcomes, we systemically examined the effect of infecting porcine AMϕ (PAMϕ) with PRRSV on their ability to produce IFN-α and TNF-α in response to stimulation with two agonists of the cytokines, namely, synthetic dsRNA [poly(I·C)] and LPS, respectively. Our results indicated that the infection of AMϕ with PRRSV does not impair the activation of the major TFs necessary for type I IFN or TNF-α gene transcription. Rather, we provide evidence that the modulation of cytokine production in PRRSV-infected AMϕ involves the actions of two endoplasmic reticulum (ER) stress sensor proteins, namely, protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1α (IRE1α).
Viral replication places a major burden on the ER to produce viral proteins, causing ER stress (23
). To cope with ER stress and maintain protein homeostasis, cells initiate the unfolded protein response (UPR), which comprises the activation of PERK, IRE1α, and a third stress sensor, namely, activating transcription factor 6 (ATF6). The UPR is aimed at promoting cell survival by reducing misfolded protein levels (25
), but it can also promote apoptotic cell death if the ER stress is not alleviated. Initially, activation of IRE1α produces cytoprotective and prosurvival responses that, despite the persistent ER stress, can become attenuated within a few hours. To reduce the burden on the ER with misfolded proteins, PERK promotes translational repression via the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). However, if the ER stress is not resolved, sustained PERK-mediated phosphorylation of eIF2α leads to C/EBP homologous protein (CHOP)-induced apoptotic cell death through various signaling pathways (26
). The infection of AMϕ with PRRSV has been shown to trigger ER stress, including the activation of IRE1α and PERK (28
). Our studies revealed that in AMϕ infected with PRRSV, NF-κB is activated by IRE1α early in infection, while eIF2α is activated by PERK late in infection. The phosphorylation of eIF2α occurring late in infection was associated with inhibited production of IFN-α and TNF-α, which could be partially reversed by an inhibitor of PERK. In contrast, early in the infection, IRE1α promoted the activation of NF-κB and enhanced TNF-α production stimulated by LPS and was abolished by an inhibitor of the kinase activity of IREα. The potential role of the UPR in promoting the production of proinflammatory cytokines is discussed as a plausible mechanism to explain the exacerbated and often lethal pneumonia that occurs during PRRSV-bacterial coinfections.
Our results showed that the infection of porcine AMϕ with PRRSV inhibits their ability to produce IFN-α in response to their stimulation with dsRNA via either cytosolic or endosomal sensors. The inhibitory effect took place even though the virus infection did not interfere with the poly(I·C)-induced phosphorylation of IRF3 (Fig. 5A
). The mechanism by which PRRSV mediates the repression of IFN-α is widely considered to act at the transcriptional level via the action of nonstructural PRRSV proteins, which seemingly have the ability to block type I IFN signaling pathways, including the activation of IRF3 (20
). The evidence supporting this notion was not generated in the context of virus infection, but rather by overexpressing single viral genes via transfection and, using reporter gene assays, examining their effects on the activity of transcription factors. This approach, however, might not necessarily reflect the events that occur during the infection of AMϕ with PRRSV. Therefore, the discrepancies between the results presented here and those from previous studies could be ascribed to the fact that we examined the influence of PRRSV infection on the signaling pathway for type I IFN production in the context of the multiphasic induction of type I IFN genes triggered by synthetic dsRNA in AMϕ. The novel contribution of our work is the analysis of the inhibitory effect of PRRSV on IFN-α synthesis in the context of PRRSV infection of its natural host cell. The performance of a stepwise analysis of the type I IFN gene induction pathway, culminating in the actual measurement of IFN-α production, enabled us to determine at what point during the virus infection of a macrophage the inhibition of IFN-α production occurred. Our results revealed that the virus infection impaired neither the activation of IRF3 and STAT1 nor the transcription of the IFNB1, IRF7, or IFNA1 gene in response to simulation of AMϕ with poly(I·C) (Fig. 4
). The activation of STAT1 and the transcription of the IRF7 gene are both components of late-phase type I IFN production, in which small amounts of type I IFN, produced early in the response, engage the type I IFN receptor (a heterodimer of IFNAR1 and IFNAR2) in an autocrine and a paracrine fashion. Binding of IFN-β/α to its receptor leads to the activation of ISGF3 (a heterotrimer of STAT1, STAT2, and IRF9), which promotes the transcription of the IRF7 gene, leading to further and robust induction of IFN-α genes and enhanced production of IFN-α (52
). Hence, it was intriguing to observe that even though the signaling pathways involved in both the early and late phases of type I IFN-α gene induction were operational, the production of IFN-α was significantly suppressed.
The results described above directed us to search for an alternative mechanism responsible for the inhibited production of IFN-α in virus-infected AMϕ. Such a mechanism should influence the late-phase type I IFN signaling pathway, should be operational >6 h after the initiation of virus infection, and should not involve the inhibition of transcriptional events necessary for TLR agonist-induced cytokine production. Like that of other RNA viruses, such as coronaviruses, the replication of PRRSV is functionally associated with the ER and places an inordinate stress on the protein-folding machinery of the organelle, thus triggering ER stress (24
). To survive ER stress, a virus-infected cell mounts the UPR, which includes activation of the stress sensor PERK, which, by phosphorylating the key translation regulator eIF2α, results in translational attenuation (24
). The results of our experiments revealed that the virus infection-induced inhibitory effect on IFN-α production in response to poly(I·C) temporally coincided with the kinetics of the appearance of phosphorylated eIF2α (Fig. 7
), which became patently manifested at >6 hpi and was accompanied by the appearance of SGs (Fig. 6E
). Since these two events are hallmarks of stalled translation (55
), it seemed likely that translation attenuation could be involved in the observed inhibitory effect of IFN-α synthesis. To further investigate this notion, we examined the effect of virus infection on the production of TNF-α in response to LPS, which yields a half-maximal response within 3 h. We reasoned that since the virus infection-induced inhibitory effect on cytokine production seemed to become operational >6 hpi, the response should not be inhibited during the first few hours after virus infection. Our results revealed that the inhibitory effect on LPS-induced TNF-α production occurs only when the virus-infected cells are exposed to the TLR4 agonist at 6 hpi. In this scenario, the bulk of the TNF-α synthesis, which occurs between 2 and 4 h after stimulation and corresponds to 8 to 10 hpi, coincides with the time at which there is an ample supply of phosphorylated eIF2α in the virus-infected cells (Fig. 10A
). On the other hand, if the stimulation of the infected AMϕ with LPS was initiated at 2 hpi, at which time the presence of phosphorylated eIF2α is negligible, there was no inhibition and a synergistic response was even observed (Fig. 9A
). Thus, we speculated that such disparate effects could be partially attributed to completion of TNF-α synthesis prior to the onset of p-eIF2α-regulated translational attenuation. Evidence of direct involvement of p-eIF2α in virus infection-induced inhibition of the TNF-α response to LPS was provided by the reduction of eIF2α phosphorylation by PERKi-treated PRRSV-infected AMϕ (Fig. 11B
) and by a significant reduction in the inhibition of TNF-α and IFN-α production in similarly treated virus-infected cells (Fig. 11C
). Together, our results indicate that the phosphorylation of eIF2α, at a late stage of the infection of AMϕ with PRRSV, involves the activation of the ER stress sensor PERK. We propose that the resulting translational attenuation is at least partially responsible for the impaired ability of PRRSV-infected AMϕ to produce IFN-α and TNF-α in response to TLR agonists. Our results are in agreement with the observations by Zhang et al. (56
), in which the poly(I·C)-induced synthesis of IFN-α by porcine monocyte-derived dendritic cells infected with PRRSV was found to be curtailed posttranscriptionally. Although the mechanism was not examined, it was proposed that it could occur via translation inhibition (57
). Raaben et al. described evidence suggesting that the UPR, triggered by the replication of murine hepatitis virus (MHV) in fibroblasts, caused host translational shutoff, as indicated by the phosphorylation of eIF2α and the formation of SGs (58
). Versteeg et al. reported that the inhibition of chemokine synthesis in fibroblasts infected with MHV was due to translational attenuation triggered by ER stress (59
). To our knowledge, this is the first report describing the involvement of the ER stress sensor PERK in the inhibition of cytokine production in response to a TLR agonist in virus-infected macrophages.
Our results also revealed that translation of viral mRNA occurs late in infection despite the marked presence of phosphorylated eIF2α (Fig. 8
). Arteriviruses, like other nidoviruses, synthesize a 3′-coterminal nested set of segmented mRNAs that contain a common 5′-end “leader sequence” (60
) from which the structural proteins are translated, presumably by cap-dependent translation (61
). Assuming that arterivirus mRNAs, like coronavirus mRNA, share important structural features with the host mRNA [such as the 5′ cap structure and 3′ poly(A) tail], they should be equally sensitive to inhibition by factors that control translational steps, including the translational attenuation resulting from the presence of p-eIF2α. Efficient translation of coronavirus mRNA in the presence of phosphorylated eIF2α has been shown to occur in cells infected with MHV (62
) and severe acute respiratory syndrome (SARS) coronavirus (63
). Possible mechanisms that have been suggested to explain the translation of coronavirus mRNA, despite the presence of phosphorylated eIF2α, include the extreme abundance of viral mRNA, which could counterbalance the effects of translational attenuation, and the use of a slightly different repertoire of translation factors (64
). It will require further studies to ascertain the mechanism by which PRRSV translates its mRNA in the presence of p-eIF2α.
Our results showed that AMϕ infected with PRRSV exhibit an increased level of phosphorylated IRE1α within 2 h after infection (Fig. 6A
), and their stimulation with LPS at such a time would result in a synergistic TNF-α response (Fig. 9A
). The phosphorylated cytosolic kinase effector domain of IRE1α interacts with the C terminus of TRAF2 (48
), ultimately resulting in NF-κB activation and upregulation of its downstream inflammatory pathways (65–67
). Direct evidence of the role of IRE1α in the virus infection-induced activation of NF-κB was provided by the observed reduction in NF-κB activation by KIRA6 (Fig. 12A
), an inhibitor of the kinase domain of IRE1α (41
). Similarly, direct evidence of the participation of IRE1α in virus infection-induced synergistic TNF-α response to LPS was provided by the abolition of the synergistic TNF-α response (Fig. 12B
) mediated by the same IRE1α inhibitor. This observation is consistent with the report that in macrophages undergoing chemically induced ER stress, the activation of IRE1α amplifies TNF-α production in response to LPS (47
In their role as sentinels against pulmonary infections (68
), the inflammatory response of AMϕ to cellular debris or to inhaled innocuous particles is relatively limited compared to a sufficiently strong proinflammatory response to respiratory pathogens that nevertheless must not compromise the vital gas exchange function of the lung (69
). However, alterations in the regulatory mechanisms that maintain a delicate balance between pro- and anti-inflammatory functional phenotypes can trigger macrophage-directed immune overreactions resulting in lung immunopathology (70
). For instance, overly robust proinflammatory cytokine responses are thought to be involved in exacerbated lung injury in bacterial coinfections with viruses, including human influenza (71
). As the most intensely studied proinflammatory cytokine, TNF-α is now considered to be a central factor in acute viral diseases, including influenza, and is prominently mentioned in cytokine storm literature (72
). In its respiratory mode, PRRSV targets AMϕ for replication and produces an interstitial pneumonia that eventually resolves (73
). Frequently, however, a PRRSV infection becomes complicated with opportunistic bacteria (74
) that commonly reside in the upper respiratory tract of pigs without producing overt disease (76
). A dual PRRSV-bacterial coinfection manifests as a severe clinical syndrome, which is characterized by an enhanced proinflammatory cytokine response, severe lung tissue damage, high morbidity, hypoxia, and often death (6
). Notably, BAL fluids collected from the lungs of animals undergoing a dual PRRSV-bacterial infection have been shown to contain relatively large quantities of TNF-α, which was considered the most likely factor responsible for the severity of the pneumonia (14
). An indication that dysregulated cytokine production in the lung could be involved in the severe respiratory syndrome observed during PRRSV-bacterial coinfections is suggested by the observation that PRRSV sensitizes the lung to respond with enhanced pulmonary proinflammatory cytokine production and severe respiratory disease upon exposure to LPS (10
). The enhanced TNF-α response of AMϕ to LPS during the early stages of PRRSV infection suggests that the activation of the stress sensor IRE1α by PRRSV infection results in a functional reprogramming of AMϕ activity toward a proinflammatory phenotype. Based on the observations presented here and those of Van Gucht et al. (10
), it seems reasonable to propose that the activation of NF-κB via the IRE1α branch of the UPR in PRRSV-infected AMϕ could dysregulate the normally moderate proinflammatory cytokine (TNF-α) response to bacterial pathogen-associated molecular patterns (PAMPs) derived from opportunistic bacteria commonly present in the respiratory tracts of conventionally raised swine (76
). In this scenario, we speculate that in the absence of PRRSV infection, AMϕ normally produce a regulated proinflammatory cytokine response to bacterial PAMPs of sufficient intensity to contain the microbes while maintaining homeostasis between the host and the resident and potentially pathogenic microbes. In the event of a respiratory infection with PRRSV, AMϕs could overreact to their exposure to bacterial PAMPs during the early stages of PRRSV infection, resulting in dysregulated production of TNF-α, promoting the development of severe inflammation and lung dysfunction. Future studies will be focused on examining the role of IRE1α in the pathogenic synergy between PRRSV and secondary bacterial pathogens. Our observations provide support to the emerging concept that the UPR directly activates proinflammatory TFs (49
) and is involved in microbe sensing by cells of the immune system (77
) and are consistent with the notion that the activation of IRE1α can act in synergy with TLR activation of proinflammatory cytokine production in macrophages (47
MATERIALS AND METHODS
The porcine AMϕ cell line ZMAC (created in our laboratory at the University of Illinois at Urbana-Champaign [UIUC]), was derived from the lungs of porcine fetuses (78
) and consists of phagocytic cells that express several surface markers characteristic of AMϕ (79
), including CD14, CD45, CD163, and CD172 (78
). The ZMAC cell line has been shown to efficiently support the growth of PRRSV (31
). Primary PAMϕ were obtained from the lavage fluid from lungs harvested from euthanized specific-pathogen-free pigs. The lung lavage fluid was obtained by infusing the trachea under aseptic conditions with 50 ml phosphate-buffered saline (PBS) per lung. The collected lavage fluid was centrifuged at 500 × g
and 4°C for 10 min. The cell pellets were washed twice with Hanks' buffered sterile saline (HBSS) and suspended in RPMI medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, Grand Island, NY, USA) and processed for freezing using dimethyl sulfoxide (DMSO). Aliquots of the resulting suspensions were stored in liquid nitrogen until further use. A single batch of primary PAMϕ was used for this study, which was chosen based on the cell population exhibiting >50% permissiveness to PRRSV. Both PAMϕ and ZMAC cells were cultured in RPMI 1640 medium containing l
-glutamine (Mediatec, Herndon, VA, USA) and supplemented with 10% FBS (Gibco), 1 mM sodium pyruvate, and 1× nonessential amino acids (Mediatec) and kept at 37°C in a 5% CO2
atmosphere. Maintenance of ZMAC cells also required the inclusion of 10 ng/ml recombinant mouse macrophage colony-stimulating factor (Shenandoah Biotechnology, Inc., Warwick, PA). MARC-145 cells (kindly provided by William Laegreid, University of Wyoming) were grown as previously described (81
Genotype 2 PRRSV strains NADC20 (82
) and FL12 (83
) were propagated in MARC-145 cells (81
). The Purdue strain of TGEV, kindly provided by Linda Saif (Ohio State University), was grown in swine testicle (ST) cells. Cell-free preparations of PRRSV were obtained from the medium overlying infected cell monolayers showing ≥80% CPE by centrifugation at 4°C and 350 × g
for 10 min. Approximately 25 ml of the clarified virus suspension was layered on top of a 3-ml cushion of 15% iodixanol (OptiPrep; Sigma-Aldrich, St. Louis, MO, USA) and subjected to ultracentrifugation at 64,100 × g
at 4°C for 3 h. The resulting virus-containing pellets were suspended in 1 ml of TNE buffer (10 mM Tris, pH 7.6, 100 mM NaCl, 1 mM EDTA). The purified virus stocks were titrated in monolayers of ZMAC cells (50% tissue culture infective dose [TCID50
]). When required, purified NADC20 virus was inactivated by exposure to short-wave (254-nm) UV light for 3 min. Loss of viability was verified by the inability of the UV light-exposed viruses to produce CPE in monolayers of MARC-145 cells. GFP-expressing PRRSV (P129-GFP virus) was kindly provided by D. Yoo (University of Illinois) (32
) and propagated in ZMAC cells. To obtain the single-step virus growth curve, ZMAC cells were infected with PRRSV strain NADC20 at an MOI of 5 to ensure a high degree of synchronous viral infection. After 1 h of infection at 37°C, the cell cultures were washed twice, and thereafter, samples of cell-free supernatants were collected at specified time intervals. The time postinfection was set at zero after the 1-h absorption. The amount of infectious virus present in samples containing PRRSV was determined in ZMAC cells according to the Reed and Muench method and expressed as TCID50
Infection, treatment of porcine AMϕ, and viability monitoring.
For viral challenge, 2 × 105
ZMAC cells were cultured in sterile 12- by 75-mm round-bottom polypropylene tubes (Corning, New York) and were either mock infected or infected with PRRSV at an MOI of 5 to obtain a synchronized infection. At the time of harvest, the cell viability was determined visually by microscopy using vital-dye exclusion. The occurrence of cellular DNA fragmentation, indicative of late stages of apoptosis, was detected by TUNEL using the DeadEnd colorimetric TUNEL system (Promega, Madison, WI). For cytokine responses, uninfected or PRRSV-infected cells cultured as described above were treated at the indicated times postinfection with either 25 μg/ml of poly(I·C) (Amersham Pharmacia Biotech Inc., Piscataway, NY) or 100 ng/ml of LPS (purified from Escherichia coli
011:B4; Sigma, St. Louis, MO) and further cultured for the indicated length of time. When indicated, the cell cultures were also treated with either 1 μM the IRE1α inhibitor KIRA6 (41
) (EMD Millipore, Darmstadt, Germany) or 1 to 2 μM the PERK inhibitor GSK2606414 (46
) (EMD Millipore, Darmstadt, Germany). As a positive control for eIF2α activation, the AMϕ cultures were treated with 2 mM DTT (Sigma-Aldrich) for 1 h. For measurement of intracellular protein status by Western blotting, 1 × 106
to 2 × 106
ZMAC cells were cultured in a 6-well tissue culture plate in a 2-ml volume. To deliver poly(I·C) into the cytoplasm, in some experiments, ZMAC cells were transfected with poly(I·C) using jetPEI-Macrophage transfection reagent (Polyplus Transfection, Illkirch, France) following the manufacturer's recommendations. Briefly, 400 μg of poly(I·C) in a 50-μl volume was mixed with 50 μl of a 150 mM NaCl solution containing 1 μl of jetPEI-Macrophage. Following a 30-min incubation at room temperature (RT), this mixture was added to a well of a 24-well plate containing 2.5 × 105
ZMAC cells in a 0.5-ml volume.
Quantitation of IFN-α and TNF-α.
Individual IFN-α-secreting cells (SC) and the presence of IFN-α in cell-free culture supernatants were detected by enzyme-linked immunosorbent spot (ELISPOT) assay and enzyme-linked immunosorbent assay (ELISA), respectively, as previously described (84
). For TNF-α detection, the same ELISA procedure was followed except that the wells were coated with 50 μl of 4-μg/ml anti-pig TNF-α monoclonal antibody (MAb) (clone103304; R&D Systems, Minneapolis, MN, USA). The captured cytokine was detected with 50 μl of 2.5-μg/ml biotin-labeled, anti-pig TNF-α MAb (clone 103302; R&D Systems). The optical densities at 450 nm (OD450
s) of triplicate wells were averaged, and the amounts of TNF-α were determined based on a curve generated from the values obtained using serial dilutions of a standard (R&D Systems). The lowest levels of detection for the IFN-α and TNF-α assays were 80 pg/ml and 120 pg/ml, respectively.
Western blot analysis.
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with a protease inhibitor cocktail (Amresco, Solon, OH, USA), and the protein concentrations of the resulting lysates were determined by using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Equivalent protein amounts of each extract (25 to 60 μg per well) were subjected to separation in an SDS-10% PAGE gel and subsequently transferred onto a 0.2-μm polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) for Western blot analysis. The membranes were incubated in blocking buffer (2% fish gelatin in TBST solution [50 mM Tris, pH 7.5, 500 mM NaCl, and 0.5% Tween 20]) at RT for 1 h. Afterward, the membranes were incubated at 4°C overnight with one of the following unconjugated primary Abs (a 1:1,000 dilution of the manufacturer's original concentration in TBST with 5% BSA): anti-IRF3 (clone D83B9; Cell Signaling, Danvers, MA, USA), anti-phospho-IRF3 (Ser396) (clone 4D4G; Cell Signaling), anti-NF-κB-p65 (3034; Cell Signaling), anti-NF-κB-p65 (Sc109; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho NF-κB-p65 (Ser536) (clone 93H1; Cell Signaling), anti-STAT1 (sc346), anti-phospho STAT1 (Tyr701) (SC-7988; Santa Cruz Biotechnology), anti-eIF2 (9722; Cell Signaling), anti-phospho-eIF2 (9721; Cell Signaling), anti-CHOP (clone L63F7; Cell Signaling), or anti-β-actin (4967; Cell Signaling). The membranes were then washed four times in TBST solution and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin (IgG) secondary Ab (sc2004; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:8,000 in blocking buffer) at RT for 1 h. After being washed again 4 times with TBST, the membranes were incubated with a chemiluminescence reagent (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) to enable detection of bound secondary Ab. Screening for the presence of a specific phosphorylated protein was always performed prior to detection of the corresponding, nonphosphorylated form on membranes that had been incubated in stripping buffer (21059; Thermo Fisher Scientific, Waltham, MA, USA) at RT for 15 min to remove any bound Ab.
RNA preparation and real-time reverse transcription-PCR.
Samples of 105
uninfected or PRRSV-infected ZMAC cells were cultured in the presence or absence of poly(I·C) for the indicated length of time. Afterward, each sample was lysed in buffer RLT, and the total RNAs were purified, DNase treated, converted into cDNA, and subjected to real-time PCR, as previously described (80
). Primers and probes for the amplification/detection of porcine IFNA1 and IFNB1 gene transcripts have been described previously (80
), whereas those associated with the amplification/detection of IRF7 and ribosomal protein L32 (RPL32) gene transcripts were designed and provided by H. Dawson (U.S. Department of Agriculture [USDA], Beltsville, MD) and are described in the DGIL Porcine Translational Research Database (http://www.ars.usda.gov/Services/docs.htm?docid=6065
). Changes in the extent of expression of the IFNA1, IFNB1, and IRF7 genes were determined by using the comparative threshold cycle (CT
) method and the formula 2−ΔΔCt
), where the RPL32 gene was used as the reference housekeeping gene.
Detection of stress granules and dsRNA in virus-infected AMϕ.
A total of 2 × 105 ZMAC cells were grown in individual wells of a Nunc LabTekII 8-well chamber slide and were either mock infected for 8 h, treated with DTT for 1 h, or infected with PRRSV strain FL12 (MOI = 5) for 4 or 8 h. Afterward, the cell monolayers were fixed in PBS containing 4% paraformaldehyde for 20 min at RT, washed with PBS, incubated with blocking buffer (PBS containing 0.3% Triton X-100 and 3% normal goat serum) for 1 h at RT, and incubated in MAb dilution buffer (PBS containing 0.3% Triton X-100 and 1% BSA) containing rabbit anti-TIAR MAb (1:200 dilution of the manufacturer's original concentration; cloneD32D3; Cell Signaling) overnight at 4°C. Afterward, the cells were washed 3 times in PBS and incubated with MAb dilution buffer containing 5 μg/ml goat anti-rabbit IgG conjugated to DyLight594 (35560; Thermo Scientific) at RT for 1 h. After being washed 3 times with PBS, the monolayers were incubated at RT in blocking buffer for 1 h and in MAb dilution buffer containing mouse anti-dsRNA monoclonal antibody (1:200 dilution of the manufacturer's original concentration; cloneJ2; Scicons, Szirák, Hungary) for 2 h, washed 3 times with PBS, and incubated with Ab dilution buffer containing 2.5 μg/ml goat anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC) (62-6511; Zymed, Life Technologies) at RT for 1 h. Afterward, the chamber was removed and the glass slide was wet mounted in antifading medium (Invitrogen, Life Technologies). Fluorescent signals were observed with an immunofluorescence microscope (DMII 4000B; Leica, Wetzler, Germany).
An unpaired Student's t
test was used to determine if significant differences existed in the cytokine gene expression or synthesis exhibited by AMϕ between treatment groups. To determine the presence of statistically significant synergism, the interaction effects between PRRSV, LPS, and the IRE1α inhibitor were tested using two-way ANOVA (51
). A P
value of <0.05 was considered statistically significant.
All experimental procedures requiring the use of animals were performed under protocols 06082 and 13064 approved by the Institutional Animal Care and Use Committee (IACUC) of UIUC. The animal care and use protocols were those followed by the Agricultural Animal Care and Use Program (AACUP), which adheres to the Federation of Animal Science Societies (FASS) Guide to the Care and Use of Agricultural Animals in Research and Teaching. The AACUP at UIUC is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC).