Bovine PI-3V is an important respiratory pathogen in young calves1,2 and is an enveloped, nonsegmented single-stranded RNA virus belonging to the genus Respirovirus in the family Paramyxoviridae. This family includes ubiquitous disease-causing viruses of infants and children, including one of the most infectious viruses known (measles), some of the most prevalent viruses known (measles, respiratory syncytial virus, types 1 to 4 parainfluenza viruses, and mumps virus), a virus that has been targeted by the World Health Organization for eradication (measles), and some newly discovered viruses (Hendra virus, Nipah virus, and metapneumovirus).
Interferons are crucial antiviral proteins induced in the early innate immune response to viral infections, including those caused by Paramyxoviridae, whether in vitro or in vivo.3,4 These cytokines not only enhance immunologic targeting of virus-infected cells by activating natural killer cell cytotoxicity and stimulating Th1 cell development, but also induce synthesis of intracellular proteins that directly inhibit viral replication by antagonizing synthesis of viral mRNA and translation of viral proteins.5 Until now, 3 IFNα- and IFNβ-responsive antiviral proteins have been considered to be involved in these processes: the MX dynamins,6 the PKR,7 and the OAS that function through RNase L.8
The MX dynamins belong to the superfamily of the large GTPases that are found in a variety of cell locations where they perform a wide array of functions including endocytosis; intracellular vesicle transport; mitochondrial distribution; and, in the case of murine, rat, and human MX proteins, interference with the replication of some viruses. They have the common characteristic that their genome consists of single-stranded negative-sense RNA molecules.6 Expression of a truncated or mutated MX isoform substantially alters the capacity of type 1 IFNs to antagonize viral replication.9 Another IFN-induced protein, PKR, is a serine-threonine kinase that is normally inactive, but is activated by binding to double-stranded RNA or other polyanions.7 Activated PKR suppresses initiation of translation via phosphorylation of eIF2α.10 Results indicate that inhibition or lack of PKR greatly increases replication of herpesviruses,11 picornaviruses,12 vesicular stomatitis virus,13 human immunodeficiency virus,14 and reoviruses.15 Oligoadenylate synthetases are a group of enzymes that catalyze the synthesis of 5′-triphosphorylated, 2′ to 5′ oligoadenylates, typically 3 or more nucleotides in length (2-5 adenylates).16 The 2-5 adenylate molecules bind with high affinity to RNase L, a dormant cytosolic endoribonuclease that upon activation by 2-5A binding catalyzes the cleavage of single-stranded mRNA and rRNA, thereby leading to inhibition of protein synthesis, thus preventing viral propagation.17 The OAS pathway is of critical importance for resistance against herpesvirus,18 flavivirus,19 and picornavirus20 infections. In the same way, disruption of the RNase L gene increases the susceptibility of mice to herpesviruses.21
The strong antiviral effect of IFNα and IFNβ against Paramyxoviridae has been commonly reported,4,22,23 including for bovine PI-3V.24,25 To our knowledge, there has been no attempt to attribute this function to any of the known IFNα- or IFNβ-induced pathways. The study reported here was undertaken to determine the contribution of MX, OAS, and PKR to the antiviral effects of type 1 IFNs against bovine PI-3V infection of Vero cells.
Materials and Methods
Experimental design—To evaluate the antiviral effect of type I IFNs on bovine PI-3V replication, Vero cells were stimulated for 20 hours with 1,000 U of IFNα/mL and subsequently infected with the virus. After removal of medium containing unadsorbed viruses, fresh medium with IFNα at the same concentration was added. Medium was harvested after 4 days incubation, and virus replication was determined by plaque assay. Confirmation of the ability of IFNα and IFNβ to interfere with the biological cycle of bovine PI-3V was also evaluated in single-cycle experiments by use of flow cytometric analysis of the proportion of immunoreactive pretreated cells versus nontreated, infected cells. In an attempt to maximize the synchronization of the process, single-cell examinations were carried out at the end of the first replicative cycle. A preliminary study therefore consisted of examining the kinetics of expressions of viral proteins, as judged from their by indirect immunofluorescence.
The role of OAS in the antiviral effect of IFNα and IFNβ was evaluated by use of transient transfection experiments in Vero cells. An empty vector was used as a negative control culture. The cells were transfected for 24 hours with the plasmid and then infected with bovine PI-3V at a multiplicity of infection of approximately 0.1. Encephalomyocarditis virus was used as a control virus for OAS activity. Because, in transient experiments, only a part of the cell population is transfected and expresses the transgene, the lack of antiviral effect may be biased. To circumvent this, single-cell evaluation of the antiviral effect of OAS was determined by examination of transiently transfected cell monolayers via double immunofluorescence and flow cytometry. To do this, Vero cells were transfected with control plasmid and plasmid coding for OAS for 24 hours and then infected for 16 hours with PI-3V.
To test the hypothesis of an antiviral effect of the MX protein against PI-3V, we first used transient transfection experiments followed by double immunofluorescence and flow cytometry analyses. Cells were transfected for 24 hours with the vector coding for huMXA or with empty vector. Double immunofluorescence and flow cytometry were performed at 16 hours after infection. A second set of experiments used 3 Vero cell clones stably expressing different amounts of huMXA. These clones were infected with the virus, and plaque assays were performed 4 days after infection.
To evaluate PKR activation, phosphorylation of eIF2α was monitored in IFNα-stimulated Vero cell monolayers infected with PI-3V or EMCV (positive control culture).
Cells, viruses, and antibodies—Madin-Darby bovine kidney (American Type Culture Collection CCL-22), baby hamster kidney 21, and Vero (American Type Culture Collection CCL-81) cells were grown and maintained in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum, 1% (vol/vol) penicillin-streptomycin, and 0.5% (vol/vol) amphotericin at 37°C in a 5% CO2–95% air humidified incubator. The bovine PI-3V and EMCV viruses were clinical isolates prepared from supernatants of infected MDBK or baby hamster kidney 21 cells, respectively; aliquots of each were stored at −80°C. Their titers were determined by use of plaque assays on Vero cells.
The bovine PI-3V antigens were detected by use of a polyclonal anti-bovine PI-3V antiserum, a monoclonal fluorescein isothiocyanate–conjugated antibody,a or a monoclonal anti-M antibody.b Human MXA protein was detected by use of polyclonal rabbit anti-huMXA antiserum. Recombinant V5 epitope–fused OAS was detected by use of mouse anti-V5 epitope antibody.c For double-immunofluorescence and flow cytometry studies, application of primary antibodies was followed by either Alexa 488–conjugated (for detection of huMXA or V5 epitope), Alexa 568–conjugated (for detection of PI-3V proteins by immunofluorescence), or phycoerythrin-conjugated antibodies (for detection of PI-3V proteins via flow cytometry). For western blotting, application of primary antibodies was followed by application of horseradish peroxidase–conjugated anti-rabbit IgG antiserum.d
Construction of expression vectors—The 2.2-kb huMXA cDNA was cloned into the EcoRI and EcoRV restriction sites of the expression vector pcDNA4/TO,c which permits constitutive high-level expression of recombinant proteins in various cell types. Full-length cDNA encoding the 69-kd form of human OAS (p69) fused to the V5 epitope was cloned in the EcoRI and HindIII restriction sites of the expression vector pcDNA4/TO.
Cytotoxicity and virus yield reduction assays— For virus yield assays, supernatants were sampled at virus-specific defined intervals and their titers determined on MDBK (for bovine PI-3V) or Vero (for EMCV) cell monolayers by use of the TCID50 method. All titers were calculated via the Reed-Muench method. The effect of recombinant human type 1 IFNe on cell viability was determined by use of an MTT cytotoxicity assay.26
Transfection assays, immunofluorescence, and flow cytometry—Transfections were performed by lipofectamine transfection.c Cells were grown to 90% confluence and transfected with 0.75 μg (24-well plates, for subsequent immunofluorescence microscopy) or 3.75 μg of vector (6-well plates, for subsequent flow cytometry) and 1 μL or 5 μL of lipofectamine, respectively, for 24 hours. Cell monolayers were then infected for 16 hours with bovine PI-3V and incubated in Dulbecco modified Eagle medium supplemented with 2% fetal calf serum.
For indirect immunofluorescence, medium was removed 16 hours after infection, and cells were washed in PBS solution and fixed with 4% (wt/vol) paraformaldehyde in PBS solution (pH, 7.4) for 30 minutes at 4°C. Cells were permeabilized in absolute methanol for 6 minutes at −20°C and blocked for 1 hour in washing buffer (1% bovine serum albumin in PBS solution) at 20°C. Cells were incubated for 45 minutes with a mixture of appropriate primary antibodies and, after 3 washing steps, incubated for a further 45 minutes with a mixture of relevant fluorescent dye–conjugated secondary antibodies diluted 1:1,000. Cells were examined with a fluorescence microscope and photographed.
For flow cytometric analysis, cells were harvested by use of a nonenzymatic method (PBS solution containing Na2EDTA) and pelleted by centrifugation at 250 × g for 5 minutes. The cells were fixed with 4% (wt/vol) paraformaldehyde in PBS solution for 30 minutes at 4°C; permeabilized in PBS solution with 0.2% (wt/vol) saponin; and blocked for 1 hour at 20°C in PBS solution, 0.2% (wt/vol) saponin, and 1% (wt/vol) bovine serum albumin. Cells were incubated for 45 minutes at 37°C with the specific primary antibodies. After 3 washing steps, the cells were incubated with fluorescent dye–conjugated secondary antibodies at 37°C. The immunolabeled cells were resuspended in 0.5% (wt/vol) paraformaldehyde–containing PBS solution and stored at 4°C until analysis. Cell suspensions were analyzed by use of flow cytometry, with gating on the forward and side scatter to exclude debris. Fluorescences were collected in FL-1 and FL-2. A minimum of 104 events were acquired and analyzed.
Generation of cells expressing huMXA protein— Vero cells were transfected by use of lipofection with the vector pcDNA4/huMXA. Zeocinc (350 μg/mL) was added to the culture medium after 48 hours, and the cells were selected for 2 weeks. After 2 rounds of cloning procedures, cells were examined by use of immunofluorescence and western blot analysis for expression of huMXA with the appropriate antibodies.
Immunoblotting—Vero cell monolayers were washed 3 times in PBS solution and harvested by trypsinization. Cellular proteins were extracted in loading buffer and separated by use of SDS-PAGE (4% to 12%). Proteins were transferred onto a polyvinylidene difluoride membrane. For huMXA detection, the membrane was incubated with mouse anti-β–actin (1:1,000) antibodies and rabbit anti-huMXA antibodies in TBS solution with 0.5× western blocking reagent. For eIF2α-phosphorylation, the membrane was incubated with the phosphorylated eIF2α (1:1,000), normal unphosphorylated eIF2α (1:1,000), and β-actin (1:1,000) specific antibodies in TBS solution with 0.5X western blocking reagent. The antibodies have been characterized by the manufacturer, and use of phosphorylated eIF2α targeting antibody has been published elsewhere.27 Following incubation with the primary antibodies, the membrane was washed in TBS-0.1% (wt/vol) Tween 20 solution and incubated with specific horse-radish peroxidase–conjugated secondary antibody (1:1,000). Immunoreactive bands were revealed on radiographic film by use of chemiluminescent technology. Densitometric analysis of immunoreactive bands obtained from 3 independent immunoblots was performed, and quantitative comparisons were made by use of the Mann-Whitney U test with P < 0.05 considered significant.
Results
IFNα and IFNβ effects on replication of bovine PI-3V—When IFN was incorporated into the medium, viral titers in MDBK cells determined from serial dilutions of the supernatants of Vero cell monolayers sampled 4 days after infection revealed a 6 log TCID50/mL reduction in virus production for all multiplicities of infection used. To rule out the possibility that the titer reduction was caused by a cytopathic rather than an antiviral effect of incorporated IFN, an MTT-based cytotoxicity reduction assay was performed. The results indicated that the treated cells were metabolically as viable as the untreated control Vero cells. Viral proteins became detectable via fluorescence microscopy 12 hours after infection. Adjacent virus-containing cells were readily detectable on and after 18 hours after infection, suggesting a secondary between-cell transmission. On the basis of these data, it was decided to examine single-cell viral protein expression at 16 hours after infection via flow cytometry. By doing so, it was confirmed that PI-3V was unequivocally inhibited in IFN-treated cells, as indicated by the reduced proportion of virus–containing cells (81.6% vs 1.6%).
Effect of OAS on replication of bovine PI-3V—Expression of OAS caused a reduction in virus production in EMCV-infected cells, but not in bovine PI-3V. The virus yield was the same in OAS-negative and OAS-positive cells. When the experiment was repeated with an enhanced green fluorescent protein–coding plasmid, no antiviral effect was seen, thus excluding a specific blockade of PI-3V by the transfection procedure. Furthermore, cells expressing both OAS and bovine PI-3V antigens were readily detected via fluorescence microscopy (Figure 1). The relative number of cells expressing viral proteins was similar between OAS expressing and nonexpressing cells as determined by flow cytometry (Figure 2).
Photomicrographs of Vero cell cultures that were transfected with pcDNA4/OAS or negative control pcDNA4/EGFP, then infected with bovine PI-3V at an multiplicity of infection of 1.0. Double immunofluorescent stain; bar = 25 μm.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photomicrographs of Vero cell cultures that were transfected with pcDNA4/OAS or negative control pcDNA4/EGFP, then infected with bovine PI-3V at an multiplicity of infection of 1.0. Double immunofluorescent stain; bar = 25 μm.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photomicrographs of Vero cell cultures that were transfected with pcDNA4/OAS or negative control pcDNA4/EGFP, then infected with bovine PI-3V at an multiplicity of infection of 1.0. Double immunofluorescent stain; bar = 25 μm.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Mean ± SD percentages of infected Vero cells transfected with pcDNA4/EGFP vector (negative control culture) or pcDNA4/OAS vector and infected with bovine PI-3V at indicated multiplicities of infection. White bars = Transduced. Black bars = Nontransduced.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Mean ± SD percentages of infected Vero cells transfected with pcDNA4/EGFP vector (negative control culture) or pcDNA4/OAS vector and infected with bovine PI-3V at indicated multiplicities of infection. White bars = Transduced. Black bars = Nontransduced.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Mean ± SD percentages of infected Vero cells transfected with pcDNA4/EGFP vector (negative control culture) or pcDNA4/OAS vector and infected with bovine PI-3V at indicated multiplicities of infection. White bars = Transduced. Black bars = Nontransduced.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Effect of huMXA on replication of bovine PI-3V— Viral antigens were readily detectable among MX-negative cells, whereas double positive cells were scarce (Figures 3 and 4). Three stable Vero cell clones were used for confirmation, 1 expressing no detectable amount of huMXA (clone 2) and 2 expressing low (clone 8) and high (clone 46) amounts, respectively (Figures 5 and 6). At a multiplicity of infection of approximately 1, clone 8 was not able to restrict replication of the virus (9.8 ± 0.6 TCID50/mL vs 9.9 ± 0.6 TCID50/mL for clones 8 and 2 respectively), whereas reduction by a factor of 10 was recorded with clone 46 (8.6 ± 0.6 TCID50/mL). At a multiplicity of infection of approximately 0.1, clone 8 repressed replication by a factor of 10 (8.7 ± 0.5 TCID50/mL) and clone 46 by a factor of 100 (7.8 ± 0.5 TCID50/mL), compared with clone 2 (10.1 ± 0.6 TCID50/mL).
Photomicrographs of Vero cell cultures that were transfected with pcDNA4/huMxA vector and infected with bovine PI-3V at a multiplicity of infection of 1.0. Double immunofluorescent stain; bar = 10 μm.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photomicrographs of Vero cell cultures that were transfected with pcDNA4/huMxA vector and infected with bovine PI-3V at a multiplicity of infection of 1.0. Double immunofluorescent stain; bar = 10 μm.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photomicrographs of Vero cell cultures that were transfected with pcDNA4/huMxA vector and infected with bovine PI-3V at a multiplicity of infection of 1.0. Double immunofluorescent stain; bar = 10 μm.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Mean ± SD percentages of infected Vero cells transfected with pcDNA4/EGFP vector (negative control culture) or pcDNA4/huMXA vector and infected with bovine PI-3V at indicated multiplicities of infection. White bars = Transduced. Black bars = Nontransduced.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Mean ± SD percentages of infected Vero cells transfected with pcDNA4/EGFP vector (negative control culture) or pcDNA4/huMXA vector and infected with bovine PI-3V at indicated multiplicities of infection. White bars = Transduced. Black bars = Nontransduced.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Mean ± SD percentages of infected Vero cells transfected with pcDNA4/EGFP vector (negative control culture) or pcDNA4/huMXA vector and infected with bovine PI-3V at indicated multiplicities of infection. White bars = Transduced. Black bars = Nontransduced.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photograph of western blot analysis of huMXA constitutive expression by 3 Vero cell clones. MW = Molecular weight marker (in kilodaltons).
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photograph of western blot analysis of huMXA constitutive expression by 3 Vero cell clones. MW = Molecular weight marker (in kilodaltons).
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photograph of western blot analysis of huMXA constitutive expression by 3 Vero cell clones. MW = Molecular weight marker (in kilodaltons).
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photomicrographs of results of expression of huMXA in the same cell clones as in Figure 5. Immunofluorescent stain; bar = 25 μm.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photomicrographs of results of expression of huMXA in the same cell clones as in Figure 5. Immunofluorescent stain; bar = 25 μm.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photomicrographs of results of expression of huMXA in the same cell clones as in Figure 5. Immunofluorescent stain; bar = 25 μm.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Effect of bovine PI-3V on PKR activation in IFN-stimulated cells—Western blotting experiments revealed quasiconstant quantities of β-actin and total eIF2α among induced and noninduced cells (Figure 7). Densiometric analysis of the phosphorylated form of eIF2α revealed a significant difference in EMCV-infected but not in bovine PI-3V–infected Vero cells primed with IFNα.
Photograph of results of immunoblotting of Vero cell monolayers infected with bovine PI-3V or EMCV at a multiplicity of infection of 1.0. Interferon A was added to Vero cell monolayers (1,000 U/mL). Immunoblotting was performed on cells extracts with anti–eIF2α-, anti–Pser51-eIF2α-, or anti–β-actin-specific monoclonal antibodies. − = Negative. + = Positive.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photograph of results of immunoblotting of Vero cell monolayers infected with bovine PI-3V or EMCV at a multiplicity of infection of 1.0. Interferon A was added to Vero cell monolayers (1,000 U/mL). Immunoblotting was performed on cells extracts with anti–eIF2α-, anti–Pser51-eIF2α-, or anti–β-actin-specific monoclonal antibodies. − = Negative. + = Positive.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Photograph of results of immunoblotting of Vero cell monolayers infected with bovine PI-3V or EMCV at a multiplicity of infection of 1.0. Interferon A was added to Vero cell monolayers (1,000 U/mL). Immunoblotting was performed on cells extracts with anti–eIF2α-, anti–Pser51-eIF2α-, or anti–β-actin-specific monoclonal antibodies. − = Negative. + = Positive.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.988
Discussion
The type 1 IFN–mediated arm of the innate immune response represents a profoundly important host defense mechanism capable of controlling the extent of virus replication, the apparent tissue tropism of the virus, and the development of disease. In the study reported here, as expected, priming of Vero cells with IFNα resulted in powerful restriction of bovine PI-3V replication. The antiviral activity of the IFNα and IFNβ systems is based on the induction and activation of protective genes (eg, PKR, OAS or RNase L, and MX) that confer resistance, inhibit virus replication, and impede virus dissemination.28
The expression, regulation, and function of the OAS and 2-5A–dependent RNase L have been characterized extensively in IFN-treated and virus-infected cells.29 The availability of cDNA clones for OAS have facilitated studies of the roles of these enzymes in processes underlying virus replication. Among the various families of viruses examined so far, the Picornaviridae had the best correlation between activation of the OAS pathway and inhibition of virus replication.30 Moreover, the so-called flavivirus–West Nile virus resistance phenotype in mice31 was finally identified as being controlled by the OAS-L1 gene, with the susceptible phenotype completely correlating with a point mutation that causes a premature stop codon.19 Without RNase L, (the downstream effector molecule of OAS), the resistance to herpes simplex virus mediated by IFNβ is lost, suggesting that the OAS pathway may also be involved in innate resistance to herpesviruses.18 In the present study, the powerful IFNα-induced resistance to bovine PI-3V was definitely not mimicked by expression of OAS. Together with a previous report32 that Sendai virus, another member of the same family, is also not inhibited, these negative results suggest that the resistance conferred by type 1 IFNs against bovine PI-3V is not attributable to implementation of the OAS–RNase L pathway.
The powerful IFNα-induced resistance to bovine PI-3V was partially mimicked by expression of huMXA. Results in the transient transfection experiments suggested a partial antiviral effect of huMXA against this virus. However, although use of stable cell lines confirmed this finding, the antiviral effect was dose dependent, with clone 46 expressing larger amounts of huMXA and blocking replication of the virus more efficiently. These results confirmed the antiviral effect of huMXA that was established previously against RNA viruses of the families Orthomyxoviridae,33 Bunyaviridae,34 Rhabdoviridae,35 Togaviridae,36 Picornaviridae,37 and Hepadnaviridae.38 As far as Paramyxoviridae are concerned, available data do not allow firm conclusions, either because conflicting results are reported (for human parainfluenza 3 virus) or because the resistance conferred is not reproducible from 1 cell line to the other (measles). Indeed, development of the IFNα-induced antiviral state against human parainfluenza 3 virus was first correlated with induction of huMXA, which led to the suggestion that it played an important role in viral inhibition.23 Later, IFNα antagonizing activity against the same virus was confirmed, but constitutive expression of huMXA had almost no effect on infectious virus production.22 Because the results gathered here for IFNα and huMXA were obtained from identical time points (16 hours after infection for bovine PI-3V), they may be interpreted as suggesting that huMXA conferred a slight resistance against bovine PI-3V but that this antagonizing effect remained far weaker than that associated with IFNα. Together with the recent report39 that an array of Paramyxoviridae (human and bovine respiratory synctial viruses, PI-3V, Sendai virus, and measles virus) were similarly resistant to the antiviral activity of the bovine homologue of huMXA, it is suggested that, overall, the MX pathway does not play an important role in IFNα-induced resistance to viruses of the Paramyxoviridae family. However, although all the data available suggest that conclusion, it remains that some cellular cofactors may decrease or exacerbate the MX-associated viral suppression, leaving the possibility that the MX pathway is only effective against paramyxoviruses when expressed in respiratory epithelial cells.
Evidence for the involvement of IFN-inducible PKR in the antiviral actions of IFNs and the control of translation in virus-infected cells comes from 3 types of analyses: study of virus replication in mammalian cells expressing PKR cDNAs,14,40 analysis of mutant mice made deficient in PKR by targeted disruption of the Pkr gene,41,42 and analysis of virus-encoded inhibitors of PKR kinase.7,43 For example, replication of EMCV,44 human immunodeficiency virus,40 reovirus,15 and vesicular stomatitis virus13 is reduced in cell culture by over-expression of PKR, and replication of herpes simplex virus is increased by targeted disruption of PKR.45 Further, in mutant Pkr0/0 mice, virulence of EMCV46 and vesicular stomatitis virus13,42 is exacerbated. Mechanistically speaking, once bound to viral double-stranded RNA, PKR becomes activated and phosphorylates eIF2α, leading to the cessation of host cell and viral translation because PKR-phosphorylated eIF2α cannot be recycled.28 In the present study, stimulation of the cells with IFNα did not result in increased phosphorylation of eIF2α upon infection, implying that the expected translation inhibitory effect of PKR does not participate in the powerful IFNα-associated suppression of bovine PI-3V. The nonphosphorylation of eIF2α implied that IFNα-induced PKR was not activated during the subsequent infection, pointing to suppression of or a lack of RNA-associated autoactivation.7,10,28 Because a number of RNA effectors of PKR function have been identified (RNA activators and RNA inhibitors), it may be suggested that bovine PI-3V does not produce the RNA intermediates necessary for PKR activation or, instead, bovine PI-3V has developed anti-PKR strategies, either by producing RNA inhibitors of PKR autophosphorylation, such as adenovirus28,43 VAI and Epstein-Barr virus EBER10; or by producing proteins that sequester double-stranded RNA activators such as influenza NS147; or that directly inhibit PKR, such as human immunodeficiency virus TAT.45
Overexpression of the MX and OAS-RNase L pathways did not result in significantly decreased viral replication, and phosphorylated eIF2α forms were not increased in IFNα-primed infected Vero cells, suggesting that the induction or activity of the major known type 1 IFNs–dependent antiviral pathways is not required for IFNα-mediated protection against bovine PI-3V in Vero cells. In addition to PKR, OAS or RNase L, and MX pathways, antiviral activity has also been attributed to other proteins induced by IFNα or IFNβ, including ADAR1,48 ISG20,49 ISG56, ISG15,50 inducible nitric oxide synthetase, and almost certainly other as-yet uncharacterized factors. The bovine PI-3V–Vero cell preparation described here will be an excellent tool to assign the powerful anti-Paramyxoviridae activity of type 1 IFNs to one or more of these pathways.
ABBREVIATIONS
| PI-3V | Parainfluenza type 3 virus |
| IFN | Interferon |
| PKR | Protein kinase R |
| OAS | 2′-5aoligoadenylate synthetases |
| eIF2α | Eukaryotic initiation factor 2 α |
| huMXA | Human MXA protein |
| EMCV | Encephalomyocarditis virus |
| MDBK | Madin-Darby bovine kidney cells |
| MTT | 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyl-tetrazolium bromide |
| TBS | Tris-buffered saline |
BioX Diagnostics, Marche, Belgium.
Institut Pourquier, Montpellier, France.
Invitrogen, Merelbeke, Belgium.
Cell Signaling Technology, Beverly, Mass.
Sigma-Aldrich, Bornem, Belgium.
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