The Amino-Terminal Domain of Bovine Viral Diarrhea
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病菌学杂志 2006年第2期
Departments of Veterinary and Biomedical Sciences, University of Nebraska—Lincoln, Lincoln, Nebraska 68583-0905
Population and Diagnostic Medicine, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 18453
Valeant Pharmaceuticals, Valeant Plaza, 3300 Hyland Avenue, Costa Mesa, California 92626
ABSTRACT
The alpha/beta interferon (IFN-/) system is the first line of defense against viral infection and a critical link between the innate and adaptive immune responses. IFN-/ secretion is the hallmark of cellular responses to acute RNA virus infections. As part of their survival strategy, many viruses have evolved mechanisms to counteract the host IFN-/ response. Bovine viral diarrhea virus (BVDV) (genus Pestivirus) was reported to trigger interferon production in infected cultured cells under certain circumstances or to suppress it under others. Our studies with various cultured fibroblasts and epithelial bovine cells indicated that cytopathic (cp) BVDV induces IFN-/ very inefficiently. Using a set of engineered cp BVDVs expressing mutant Npro and appropriate controls, we found that the IFN-/ response to infection was dependent on Npro expression and independent of viral replication efficiency. In order to investigate whether the protease activity of Npro is required for IFN-/ antagonism, we engineered Npro mutants lacking protease activity by replacement of amino acid E22, H49, or C69. We found that E22 and H49 substitutions abolished the ability of Npro to suppress IFN, whereas C69 had no effect, suggesting that the structural integrity of the N terminus of Npro was more important than its catalytic activity for IFN-/ suppression. A catalytically active mutant with a change at a conserved Npro region near the N terminus (L8P) in both BVDV biotypes did not antagonize IFN-/ production, confirming its involvement in this process. Taken together, these results not only provide direct evidence for the role of Npro in blocking IFN-/ induction, but also implicate the amino-terminal domain of the protein in this function.
INTRODUCTION
The genus Pestivirus comprises an important group of animal pathogens that includes agents such as Bovine viral diarrhea virus (BVDV) and Classical swine fever virus (CSFV). Based on their molecular phylogenies, as well as physical, chemical, and biological properties, the genus Pestivirus is classified in the family Flaviviridae (74). The pestivirus genome consists of a single RNA molecule approximately 12 kb in length and with positive polarity (14, 43). The enveloped virions deliver the genomic RNA to target cells by fusion with endocytic membranes (21). Pestiviruses replicate exclusively in the cytoplasm, generally without inducing death of the infected cultured cells, i.e., most pestiviruses are noncytopathic (ncp) (27).
Much of the economic impact of pestivirus infections results from their tropism for the fetus. Fetal infection with ncp BVDV during the first trimester of gestation results in the birth of calves that are persistently infected and immunotolerant to the viral antigens. These newborn calves develop into apparently healthy animals, which remain viremic for life. The ncp BVDV virus pool within persistently infected animals often gives rise to cytopathic (cp) mutants, which cause a fatal syndrome known as mucosal disease (8, 11, 46, 47, 49, 54). Cp BVDV variants induce apoptosis in cultured cells (29, 30, 32, 76, 81). It is thought that immunologic tolerance allows unchecked replication of these cp BVDV variants, triggering the pathogenic cascade associated with mucosal disease (9). The biochemical hallmark of cp variants is the production of high levels of NS3 as a free protein after the early phase of infection (17, 39, 57). In contrast, ncp BVDV variants produce largely NS2-3 and small amounts of NS3 during the early phase of infection (39). The pathogenesis of the fatal mucosal disease syndrome is poorly characterized; however, excessive stimulation of innate immune responses may play a significant role. Alpha/beta interferons (IFN-/) are powerful cytokines which regulate cells from most organ systems in the body with regard to their permissiveness to viral infection and many other specialized functions, e.g., differentiation of dendritic cells (25, 50, 55, 70).
The production of IFN-/ in virus-infected cells is transcriptionally regulated (45). IFN-/ gene transcription is activated by multiple signals, the best characterized being viral infection, in which signaling is thought to actually be mediated by double-stranded RNA (dsRNA) (26, 38, 79). Viral infections, particularly those caused by RNA viruses, involve the production of cRNA molecules in the form of extended dsRNA structures. dsRNA, as well as endoplasmic reticulum stress, oxidative stress, and additional uncharacterized viral signals, activates a cascade of events that converge to activate several transcription factors, such as NF-B and ATF-2/c-jun, as well as interferon regulatory factor 3 (IRF-3) and IRF-7 (2, 4, 35, 38, 68, 69, 79). The resulting enhanceosome is responsible for a surge of IFN-/ gene transcription, leading to translation and secretion. IFN-/ molecules bind interferon receptors (IFN-/R) present in virtually all nucleated cells of the body to activate an antiviral program in the target cells. To counter this antiviral response of host cells, many viruses have evolved effectors that actively interfere with the IFN-/ system or with the downstream interferon-stimulated gene products that mediate the antiviral state (7, 23, 24, 28, 48, 51, 73, 78, 82). Viruses that target the cellular IFN-/ system have two general modes of action: they either suppress the production of IFN-/ or interfere with the IFN-/R signaling pathway (7, 24, 67). Previous studies have shown that infection of cultured cells with cp BVDV triggers IFN-/ production in macrophages and in some nonmyeloid tissue cultures, whereas no IFN-/ is produced by ncp-BVDV-infected cells of any type (1, 6, 56, 66). However, both cp and ncp BVDVs have been shown to block the function of IRF-3, which is required for IFN-/ transcription (5, 6). Thus, cp-BVDV infection is reported to either induce or suppress IFN-/ in infected cells. The Npro and Erns proteins have been reported to participate in IFN-/ suppression during CSFV infection (34, 41, 64). The Npro protein is dispensable for the growth of BVDV and CSFV in cell culture, but deletion mutants are growth impaired in cell culture (40, 71). The growth impairment of CSFV Npro deletion mutants has been recently linked to interferon antagonism (64). Recently, it was demonstrated that the Npro protein of CSFV can impair IRF-3 activity (33, 41). The genetic analysis of Npro mutants of pestiviruses is complicated by the fact that changes in the RNA sequence that encode this protein can affect polyprotein translation and cleavage of the capsid protein, as well as host cell IFN-/ responses. The RNA sequences encoding Npro that are adjacent to the internal ribosome entry site (IRES) element modulate translation of the viral polyprotein (53, 60). In this report, we show that cp-BVDV infections do not induce detectable levels of IFN-/ and that the phenotype depends on Npro expression. We also found that antagonism to IFN-/ production by viral infection requires the N-terminal domain of Npro, whereas its endoproteinase function seems dispensable for this purpose.
MATERIALS AND METHODS
Cells and viruses. Bovine testicle cells express epithelial markers and were derived as described previously (18). NCL1 bovine uterine cells are also epithelioid (E. J. Dubovi, unpublished data), whereas CRIB cells resistant to BVDV infection (20) were derived from MDBK cells. All cells were grown in minimum essential medium (MEM) supplemented with 10% equine serum (ES; HyClone) and 1x penicillin-streptomycin antibiotics (Life Technologies). The NCL1-ISRE-Luc-Hygro cell line (L. Gil and R. Donis, unpublished data) is a stable bovine cell line selected for integration of the luciferase reporter under transcriptional control of an IFN-stimulated response element (ISRE); it was cultured in the same medium supplemented with 300 μg/ml hygromycin (A. G. Scientific, Inc.).
pNADLp15 (cp NADL), N-dINS (ncp NADL), N-H/B, and 5B-1HR are modified viruses derived from a plasmid containing the full-length cDNA of the BVDV strain NADL genome, as described elsewhere (3, 40, 76). The mutant 5B-1HR was derived by reverse genetics from the NADL strain of BVDV by insertion of a 19-amino-acid epitope tag at position 704 of NS5B (insertion of 57 nucleotides at position 12303) (3). Vesicular stomatitis virus (VSV) New Jersey serotype was obtained from F. Osorio (University of Nebraska).
Antibodies. BVDV Npro polyclonal antibody (kindly provided by X. Cao, YC Inc., Boston, MA), NS3 monoclonal antibody 20.10.6 (15), horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) antibody (Sigma) and HRP-conjugated anti-mouse IgG antibody (Sigma) were used for Western blot analysis. The goat anti-BVDV polyvalent antiserum (VMRL Inc.) and secondary recombinant HRP-conjugated protein G (Zymed) were used for focus assays.
Virus preparation. All experiments aimed at measuring IFN-/ responses to virus infection were performed using pelleted virus preparations to infect cells. This step removed IFN-/ and other cytokines that could be present in the cell culture fluids harvested to prepare BVDV stocks. Virus stocks were produced by inoculating NCL1 cells with seed virus at an input multiplicity of infection (MOI) of 1. The cell culture fluid was harvested at 24 h postinfection (p.i.) and clarified by low-speed centrifugation (2,000 x g) for 30 min. The clarified culture fluid was centrifuged at 100,000 x g for 2 h. The supernatant was discarded, and the virus-containing pellet was drained, resuspended in a small volume of MEM with 5% horse serum, and frozen in aliquots. Stocks used in these studies had titers of 7.5 to 7.7 log10 median tissue culture infective doses/ml (36).
Virus infection. Bovine cells were infected with pelleted virus stocks at an MOI of 10 in MEM containing 5% equine serum. After an absorption period of 1 h, the inoculum was removed and replaced with fresh medium.
Virus growth kinetics. To determine the viral replication efficiency, bovine uterine cells were infected with each virus at an MOI of 0.3. After 1 h of incubation at 37°C, the inoculum was removed and the cell monolayers were washed three times with MEM. Fresh MEM containing 5% ES was added, and the cells were incubated at 37°C. The supernatants were harvested at different times after infection, and their infectious titers were determined by serial dilution.
Plaque assay. Bovine testicle cell monolayers were infected with serial 10-fold dilutions of the viruses. Following 1 h of adsorption at 37°C, the inoculum was removed, and the cells were overlaid with 0.8% agarose in MEM plus 5% equine serum and incubated at 37°C. After 4 days, the cells were fixed and stained with 0.05% crystal violet in 10% formalin.
Focus assay. The plaque assay was done as indicated above. After 4 days, the cells were fixed with cold 30% acetone in phosphate-buffered saline (PBS) and air dried. The monolayers were sequentially incubated for 90 min with goat anti-BVDV polyvalent antiserum, followed by a 60-min incubation with recombinant HRP-conjugated protein G. Both were diluted in PBS containing 0.01% Tween 20 and 0.5 M NaCl. Bound conjugates were revealed with the peroxidase substrate 3-amino-9-ethylcarbazole (Sigma).
Construction of BVDV mutants and sequence analysis. BVDV mutants were generated using a reverse genetics system established previously (42, 75). The introduction of mutations was confirmed by reverse transcription (RT)-PCR and sequence analysis. Descriptions of constructs are presented below. Viral RNA was extracted using a commercial lysis solution and resin kit (RNAeasy; QIAGEN). RT-PCR was carried out using a one-step RT-PCR kit (QIAGEN). cDNA products were purified using QIAquick PCR purification (QIAGEN). Sequencing reactions were performed using the ABI BigDye terminator cycle-sequencing kit with products resolved on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The sequences of primers used for amplification and sequencing are available upon request. DNA sequence analysis was performed using version 10 of the Genomics Computer Group sequence analysis package (16).
RNA transcription and electroporation. RNA transcripts were prepared using a MEGAscript in vitro transcription kit as recommended by the manufacturer (Ambion). The concentration of purified RNA was determined by staining with RiboGreen RNA quantification reagent (Molecular Probes), as described previously (77).
The in vitro-transcribed RNAs were transfected into bovine uterine cells by electroporation as described previously (3). Briefly, bovine uterine cells were trypsinized, washed two times with MEM, and resuspended at 2 x 107 cells/ml in cytomix (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4, pH 7.6, 25 mM HEPES, pH 7.6, 2.0 mM EGTA, 5.0 mM MgCl2) plus 2.0 mM of ATP and 5.0 mM of glutathione (72). Unless otherwise indicated, 3 μg of transcribed RNA was mixed with 0.1 ml of cells in suspension and immediately pulsed with a BTX-600 ElectroSquarePorator (320 V; 100 μF; 48 ). The electroporated cells were diluted in 10 ml of MEM-ES and plated on tissue culture dishes.
RNA infectivity determination by infectious-center assay. An infectious-center assay was used to quantify the specific infectivity of in vitro-transcribed RNA. Tenfold dilutions of electroporated bovine uterine cells were added to preseeded monolayers of MDBK cells grown to 60% confluence. Following 4 h of incubation, the cells were washed and overlaid with 0.8% agarose in MEM-5% equine serum. The plates were incubated for 3 days at 37°C, and infectious centers were visualized and counted by staining the plaques as described above.
Northern blots. Bovine testicle cells (106 cells; 60-mm dish) were infected with purified BVDV at 10 PFU/cell. At 1 h postinfection, the cell monolayers were washed with 5 ml of PBS, followed by the addition of 5 ml of MEM containing 5% ES. The total RNA from bovine uterine cells was prepared by using an RNeasy total-RNA kit (QIAGEN). Five micrograms of total RNA was separated in a formaldehyde-containing agarose gel, transferred onto a nylon membrane (Hybond N+; Amersham Biosciences), and probed by hybridization to a DNA fragment spanning nucleotides 6044 to 6432 of BVDV-NADL labeled with [-32P]dATP by a random priming method (Prime-a-Gene Labeling System, Promega). The membranes were washed twice in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS) at room temperature and once in 0.1x SSC-0.1% SDS at 60°C for 30 min and exposed to film (X-Omat; Kodak). The blots were rehybridized with GAPDH (glyceraldehyde phosphate dehydrogenase) probe to ascertain the lane loading and transfer efficiency. Scanning densitometry was achieved by PhosphorImager analysis with ImageQuant software (Molecular Dynamics). The signal intensities from BVDV-specific bands were normalized by comparison to the signal from the cellular housekeeping gene GAPDH mRNA.
Western blots. Proteins were separated by SDS-polyacrylamide gel electrophoresis (10% or 12% acrylamide) and transferred onto a nitrocellulose membrane (HybondTM; Amersham Life); the membrane was blocked with 5% (wt/vol) dried skim milk in PBS containing 0.005% Tween 20. For the detection of the Npro protein, membranes were probed with polyclonal antibody specific against the Npro protein of BVDV, followed by incubation with a secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody. The NS3 or NS2-3 protein of BVDV was detected by using the monoclonal antibody 20.10.6, which is specific for the pestivirus nonstructural protein NS3, and the bound antibodies were detected by horseradish peroxidase-conjugated anti-mouse IgG antibody (15). The membranes were rinsed five times in PBS containing 0.005% Tween 20 after primary and secondary antibodies. The membrane-bound immune complexes were revealed by the enhanced-chemiluminescence detection system (Amersham), and images were captured on X-ray film.
Reporter plasmids and electroporation. pIFN-beta-CAT expresses choramphenicol acetyltransferase (CAT) under the control of the IFN- enhancer element (19), pISRE-Luc expresses firefly luciferase under the control of an interferon-stimulated response element (Stratagene) (31), and pRL-SV-40 (Promega) expresses Renilla luciferase under the transcriptional control of the simian virus 40 promoter element. Reporter plasmids were delivered into cells by electroporation of DNA into cells in suspension. Briefly, bovine uterine cells (90% confluent) were trypsinized, washed twice with MEM, and resuspended at 2 x 107 cells/ml in cytomix (72). Unless otherwise indicated, 3 μg of reporter DNA plasmid was mixed with 0.1 ml of cells in suspension and immediately pulsed with a BTX-600 ElectroSquarePorator (320 V; 100 μF; R3) as previously described (3). The electroporated cells were diluted in 12 ml of MEM-ES and seeded (1 ml per well) into 12-well plates. The plates were incubated for 4 h at 37°C, after which the medium was replaced with fresh medium. At 24 h postelectroporation, the cells were mock infected or infected with BVDV at an MOI of 10. At the indicated times after infection, cells were lysed in reporter lysis buffer (Promega). CAT assays were performed using 14C-labeled chloramphenicol and n-butyryl coenzyme A, following the CAT assay kit instructions (Promega). The luciferase assay was performed by using the dual-luciferase (firefly and Renilla) assay system (Promega) according to the manufacturer's protocol. Expression levels were normalized for transfection efficiency by dividing the firefly luciferase or CAT values by those of Renilla luciferase. The relative expression of reporter enzyme (change [n-fold]) was calculated by dividing each test sample into the mock-infected control value.
IFN-/ bioassay. The production of IFN-/ in BVDV-infected cells was determined according to the method of Rubinstein et al. (63) with some modifications. Bovine cells were infected with purified BVDV or BVDV mutants at an MOI of 10. At 24 h postinfection, the medium was clarified by centrifugation. In order to inactivate BVDV and other cytokines present in the samples, cell culture supernatants were adjusted to pH.2 with a 2 M hydrochloric acid solution. After 24 h at 4°C, the pH was restored to 7 with a 2 M sodium hydroxide solution. Serial dilutions in MEM-5% ES were added to 96-well plates and overlaid with 0.1 ml of MEM-5% ES containing CRIB cells at a concentration of 3 x 105 cells per ml. After incubation in a moist chamber at 37°C for 16 h, CRIB cell monolayers were infected with VSV at an MOI of 0.1. After incubation for an additional period of 24 h, the monolayers were fixed and stained with crystal violet and the IFN titer was determined as follows. The number of IFN equivalent units in the experimental samples that conferred 50% protection from the cytopathic effect of VSV was determined by comparison with samples analyzed in parallel using titrated units of recombinant bovine IFN- as a standard (kindly provided by Lorne Babiuk, VIDO, Saskatchewan, Canada).
IFN reporter gene assay. The IFN-/ activity was also measured by using the NCL-1-ISRE-Luc-Hygro cells, a stable bovine cell line with the ISRE-Luc reporter cassette integrated into its genome (Gil and Donis, unpublished). Briefly, 1.5 x 105 cells were seeded into a 12-well plate and cultured at 37°C. After incubation in a moist chamber at 37°C for 12 h, the medium was replaced with 0.5 ml of test sample and incubated for 8 h. Cells were harvested and lysed in 100 μl of passive lysis buffer (Promega), and the firefly luciferase activity was measured as described above. This assay is sensitive to bovine IFN-/ and detected IFN-/ levels as low as 5 IU/ml (Gil and Donis, unpublished).
PINBA. Bovine testicle cell were infected with ncp BVDV (MOI = 1) and passed twice before being used for the poly(I · C) for ncp BVDV assay (PINBA). The PINBA was performed as described previously (44, 61) with some modification. Bovine testicle cells, uninfected or persistently infected with ncp BVDV, were seeded into a 96-well plate at a concentration of 3 x 104 cells/well and incubated for 6 h at 37°C. After incubation, the medium was removed and 50 μl of fresh medium per well with or without 50 μg of poly(I · C) per ml was added. Twelve hours later, the cell monolayers were challenged with VSV infection at an MOI of 0.1. Following overnight incubation, the monolayers were stained with crystal violet to better visualize the VSV cytopathic effects.
RESULTS
IFN-/ secretion by cells infected with Npro mutants of BVDV. The presence of a nonstructural protein at the 5' end of the polyprotein is a defining feature of pestiviruses. This polypeptide, termed Npro, is an unconventional protease capable of autocatalytic cleavage at its own C terminus (65, 80). To characterize the role of the Npro protein, a series of Npro BVDV mutants were used (Fig. 1). Two of the Npro mutants were deletion mutants, one (N-dNpro) in which the Npro protein was completely deleted and the other (N-H/B) in which Npro was replaced with a heterologous autocatalytic cleavage cassette derived from the hepatitis C virus (HCV) NS3 protease domain. The two Npro deletion mutants were previously found to be phenotypically similar (40). The growth curve of a mutant virus lacking Npro showed that the virus progeny levels were 10-fold lower than those of wild-type (wt) virus at each of the time points (Fig. 2B) (40). This observation was correlated with an 3-fold reduction of specific infectivity of the RNA compared to that of the wt virus (Fig. 2A). The infectivity of the RNA, growth kinetics, plaque size, and RNA accumulation revealed that the N-H/B chimeric virus is similar to the N-dNpro mutant in its replication efficiency (Fig. 2A to D). The third Npro mutant, termed N-Npro18GFP, contains the jellyfish green fluorescent protein (GFP) gene cloned within the N-terminal region of Npro, between codons 18 and 19 (Fig. 1). Although the autocatalytic protease activity of this Npro chimera remained intact (data not shown), the virus formed small plaques like the other two mutants (Fig. 2C). The analysis of cellular responses to infection by Npro deletion and chimeric mutants is complicated by the fact that Npro mutant viruses have reduced replication efficiency and, consequently, delayed induction of apoptosis compared to the wt virus. Thus, to test the hypothesis that increased cytokine production and secretion in cells infected by Npro mutants is not attributable to a decreased impairment of host cell metabolism compared to that of cells infected by wt virus, we also developed and characterized an isogenic BVDV mutant with an altered NS5B polymerase gene, termed 5B-1HR. Comparative analysis of this mutant with the Npro deletion viruses revealed that it displays a degree of replication deficiency similar to that observed with Npro mutants (Fig. 2C and D).
We used our panel of mutant chimeric and mutant cp BVDVs and several bovine cell lines to assess the effects of viral infection on IFN-/ production. To this end, we first used the VSV bioassay to measure IFN-/ levels in the culture media of infected cells. No detectable IFN was present in the culture media of cells infected with wt NADL or the ncp derivative N-dINS, whereas media from cells infected with Npro mutants showed high levels of IFN-/ (Table 1). Although the levels of IFN-/ varied among cell lines, three different cell lines with diverse origins—uterine, testicular, and renal—showed the same pattern of response to infection, suggesting that the consequence of viral infection was reproducible (Table 1). Bovine cells lacking BVDV receptor function (CRIB) inoculated with Npro mutant viruses showed no detectable IFN-/ in the medium, suggesting that induction of IFN-/ production was dependent on viral infection and did not involve signaling through plasma membrane receptors (20). CRIB cells were otherwise responsive to another IFN-/ inducer, poly(I · C) (Table 1).
We used the 5B-1HR linker insertion mutant, which is phenotypically very similar to the mutants with Npro deleted with regard to plaque size and viral RNA accumulation, to determine whether the cellular IFN-/ response was a correlated with viral replication efficiency or Npro expression (Fig. 2C and D). We found that mutant 5B-1HR-infected cells responded like the wt-virus-infected cells: no detectable levels of IFN-/ were present in the culture medium (Table 1). Thus, high levels of IFN-/ secretion after infection with growth-impaired mutants are correlated with a lack of intact Npro expression and not with reduced cytopathogenicity for the infected cells.
IFN-/ and ISRE gene transcription in BVDV-infected cells. The high levels of IFN-/ released into the media of cells infected with Npro deletion mutants could result from transcriptional or posttranscriptional regulation events. To investigate these possibilities, cells were transiently transfected with human IFN--CAT reporter or ISRE-Luc reporter plasmids and subsequently infected with wt or Npro mutant viruses. Cells infected with the Npro mutants showed 40- to 70-fold- and 80- to 145-fold-increased levels of activation of the IFN- and ISRE promoters at 20 h p.i. compared to the wt virus or the 5B-1HR mutant (Fig. 3A and B). Cells infected with N-dINS (ncp NADL) and wt NADL showed background levels of IFN- promoter activation identical to those of mock-infected cells, in agreement with our earlier data (Table 1). These results implicate IFN-/ gene transcription regulation as the major determinant of secreted cytokine levels and reveal the possible autocrine or paracrine action of the secreted IFN-/ in the activation of the ISRE enhancer element.
Role of the catalytic domain of Npro in IFN-/ antagonism. Recent studies showed that the HCV NS3/4A serine protease blocks the phosphorylation of IRF-3, a key cellular antiviral signaling molecule (22). To investigate the importance of the proteinase activity of Npro in IFN-/ antagonism, we utilized a reverse genetics approach targeting residues E22, H49, and C69, individually known to be essential for the catalytic activity of Npro in pestiviruses (65). However, mutations that abolish the proteinase activity of Npro are lethal due to improper maturation of the capsid protein, which in turn prevents the formation of infectious virions (40). To overcome this problem, we engineered a virus whose genome consisted of the N-H/B genome (Fig. 1) with the Npro protein cloned upstream of HCV NS3 protease (Fig. 4A). The restored virus, termed N-Npro-H/B, produced plaques slightly larger than those of the parental virus N-H/B (2.45 ± 0.44 mm in diameter versus 1.7 ± 0.29 mm), which we hypothesized stemmed from the restoration of Npro function (Fig. 4B). To determine whether the improved growth phenotype of N-Npro-H/B involved IFN-/ inhibition, we analyzed the production of IFN-/. No detectable IFN-/ activity was present in the medium of bovine cells infected with N-Npro-H/B, confirming previous observations that the Npro protein has IFN-/ antagonist properties (Fig. 4C). Most importantly, the endoproteolytic activity of Npro in this genome is predicted to be dispensable for release of the capsid protein C. Therefore, the genome was deemed suitable for engineering Npro catalytic mutants. Mutants of residues previously described as critical for the protease activity of Npro were engineered in the N-Npro-H/B infectious clone, giving rise to the following viable viruses from full-length genomes: N-Npro-H/B-E22L, N-Npro-H/B-H49V, and N-Npro-H/B-C69A (Fig. 5A). The consequences of these mutations for Npro endoproteinase activity were determined by tracing Npro in infected cell lysates by Western blotting with a specific polyclonal antibody (Fig. 5B). Npro expression was readily detected as a 20-kDa band in blots from lysates of cells infected with wt and N-Npro-H/B. In contrast, the three protease mutant viruses lacked the 20-kDa protein and expressed only a fusion protein of approximately 40 kDa, comprising the mutated Npro and the HCV protease. Interestingly, the cleavage of Npro by N-Npro-H/B is incomplete, and substantial amounts of the Npro-HCV protease fusion protein are detected. Regardless of the cause of the reduced catalytic function of Npro as an endoproteinase, the plaques formed by the parental virus N-NproH/B and the N-NproH/B-C69A mutant were comparable in size (1.3 ± 0.3 mm and 1.4 ± 0.3 mm in diameter, respectively), suggesting similar replication efficiencies. However, the plaques produced by the N- NproH/B-E22L and N- NproH/B-H49V mutants were smaller (0.6 ± 0.19 mm and 0.76 ± 0.29 mm, respectively) (Fig. 5C).
We next analyzed IFN-/ induction in the culture media of cells infected with each protease mutant (Fig. 5D). Interestingly, cells infected with the E22 and H49 mutants induced high levels of IFN. In contrast, the C69 mutant induced background levels of IFN-/, suggesting that the catalytic endoproteinase activity of Npro is dispensable for the IFN-/ antagonist function. In this context, the lack of IFN-/ antagonism of mutants E22 and H49 could be explained by disruption of a hypothetical IFN-/ regulatory domain located near the N terminus of Npro.
A conserved domain at the N terminus of Npro is essential for IFN antagonism. Alignment of multiple pestivirus Npro sequences revealed a 5-amino-acid sequence motif (LLYKT; codons 8 to 12) near the amino terminus of the protein as the largest conserved block in the region. In order to study the contribution of this conserved domain to IFN antagonism, we constructed a mutant viral genome termed N-L8P, in which the highly conserved leucine at position 8 was changed to proline. The N-L8P virus formed considerably smaller plaques than the wt virus (1.9 ± 0.4 mm versus 2.9 ± 0.95 mm in diameter), resembling the plaques formed by the viruses with Npro deleted (Fig. 6A). This was correlated with an 68% reduction in progeny yield at various times after infection (Fig. 6D). The stabilities of Npro and its endoproteinase were examined by Western blot analysis of the steady-state levels of the protein in infected cells. The Western blots of bovine testicle cells infected with the N-L8P mutant revealed that Npro expression levels and endoproteinase activity were similar to those of the parental viruses (Fig. 6B). Interestingly, culture medium from cells infected with N-L8P contained high levels of IFN-/, as indicated by a 60-fold increase in luciferase expression in reporter cells relative to controls (Fig. 6C). Thus, the impaired growth of N-L8P was correlated with the induction of IFN-/ production in the infected cells. Similarly high levels of IFN-/ were detectable in the medium from cells infected with N-dNpro, which yielded a 56-fold increase of reporter gene expression. These data indicated that a single amino acid change (L8P) within the N terminus of Npro resulted in a virus that is phenotypically identical to the Npro deletion mutants with regard to IFN-/ induction.
To increase our understanding of the IFN-/ antagonistic function of the BVDV Npro protein, we create the L8P ncp mutant virus. Figure 7A shows the focus-forming phenotypes of N-dINS (the ncp NADL parental virus) and N-dINS-L8P (the ncp L8P mutant virus); both viruses displayed focus formation with similar sizes (2.3 ± 0.7 mm versus 2.2 ± 0.5 mm in diameter, respectively). Levels of NS2-3 expression were examined by Western blot analysis of cell lysates infected at an MOI of 5 PFU/cell and collected at 18 h p.i. (Fig. 7B). Unsurprisingly, the levels of NS2-3 and NS3 expression were higher for the wt NADL than N-L8P. However, uncleaved NS2-3 levels were similar for N-dINS and N-dINS-L8P. IFN-/ analysis (Fig. 7C) using an ISRE luciferase reporter cell line indicated that supernatants from mock-infected cells or cells infected with wt NADL and N-dINS did not contain IFN-/ activity. Interestingly, supernatants from cells infected with N-dINS-L8P showed a very low level of IFN, which resulted in a 1.5-fold increase in luciferase expression. In contrast, supernatant from N-L8P-infected cells induced a 58-fold increase in luciferase expression. These results suggest that N-dINS-L8P is a poor IFN inducer in cultured cells, and this characteristic might be due to the low levels of viral-RNA accumulation present in ncp-BVDV-infected cells (76).
Next, we tested whether N-dINS-L8P virus could block IFN-/ induction by dsRNA treatment. For this purpose, we used the PINBA previously described (44, 61). The results of this experiment demonstrated that, unlike N-dINS, cells persistently infected with N-dINS-L8P virus are not able to inhibit IFN-/ induction by dsRNA and consequently were nonsusceptible to VSV infection (Table 2). Taken together, these results strongly suggest that the N-terminal domain of the BVDV Npro protein plays a critical role in IFN-/ suppression.
DISCUSSION
We exploited a reverse genetics approach to analyze the role of Npro in virus-host interactions. We first studied two BVDV mutants with Npro deleted and one Npro mutant derived from the same infectious clone but structurally distinct at the junction between the IRES element and the polyprotein open reading frame. We also analyzed a mutant with intact Npro that is phenotypically similar to the mutants with Npro deleted with regard to replication efficiency and RNA accumulation. Contrary to the prevailing notion that wt NADL infection induced IFN-/, we found that, at least in our systems, infection with wt NADL was indistinguishable from mock infection or ncp BVDV infection. Assay sensitivity cannot explain these differences, because we used a highly sensitive reporter cell line to detect IFN-/ in concentrations as low as 5 IU/ml (data not shown). Because these observations were the same in different cell lines, we cannot attribute the failure of wt NADL to induce IFN-/ to host factor variability. Analysis of IFN-/ in the supernatants of bovine cells infected with different cp BVDV strains detected only low levels of IFN-/, which suggests that cp BVDV is a poor IFN-/ inducer (data not shown).
In contrast to the wt NADL, mutants with Npro deleted elicited very high levels of IFN-/, regardless of the type of bovine cell line used. The unrestrained IFN-/ production triggered by infection with an Npro deletion mutant appeared to contribute significantly to the reduced growth of these mutants. The reduced efficiency of IRES function in the Npro deletion mutants might further contribute to the replicative impairment.
The importance of Npro for IFN-/ suppression was made clear by the restoration of Npro to the N-H/B deletion mutant virus. N-Npro-H/B showed no IFN-/ induction and produced plaques larger than those of the parental virus (N-H/B), which induced IFN-/ production. Interestingly, transient expression of Npro by plasmid transfection did not result in functional complementation; the growth of the Npro deletion mutants could not be rescued, presumably because of inefficient Npro expression in transfected bovine cells. Previous reports implicating a viral proteolytic activity in the suppression of host IFN-/ response by HCV NS3 prompted us to examine whether the Npro endoproteinase has an analogous function (22). Mutagenesis of Npro effectively disabled its catalytic activity, confirming the findings of Rumenapf et al. (65). One of the catalytically inactive Npro mutants (C69) showed efficient suppression of IFN-/ induction, strongly suggesting that the catalytic activity of Npro is dispensable for this function, although the unlikely possibility that an undetected residual protease activity is responsible for IFN-/ suppression cannot be ruled out. However, the two other mutants (E22 and H49) with a catalytically disabled Npro lost IFN-/-antagonistic activity. Although this could point to the importance of the catalytic activity of Npro for IFN-/ suppression, it is also possible that the structural changes that disable catalysis also disrupt an overlapping domain involved in IFN-/ antagonism. The second hypothesis is consistent with the identification of L8 as a critical amino acid in a conserved region of the N-terminal domain. Lack of IFN-/ suppression by mutants L8P, E22L, and H49V suggests that the N-terminal 49 amino acids of Npro are essential for the suppression of IFN-/ production in infected cells. In addition, the cloning of N-dINS-L8P is consistent with the IFN-/-antagonistic properties of the N-terminal domain of the Npro protein and demonstrated that the L8 mutation in the ncp NADL strain abolished the ability of the ncp virus to inhibit IFN-/ induction by dsRNA treatment. Surprisingly, cells infected with N-dINS-L8P did not induced high levels of IFN-/ production as expected. One reason for this unexpected outcome of N-L8P versus N-dINS-L8P might be related to different amounts of dsRNA being formed during replication. Previous data showed that the amount of RNA present in cells infected with cp BVDV vastly exceeds that in cells infected with ncp biotypes (76). Therefore, it is tempting to speculate that a large amount of this viral RNA is implicated in the IFN-/-inducing activity of the N-L8P virus.
The identification of Npro as a potential suppressor of IFN-/ production by BVDV-infected cells provides a candidate target for intervention, since it is becoming increasingly clear that this function is part of the essential machinery of animal viruses. HCV was recently shown to have developed a mechanism to suppress IFN-/ transcription activation by way of its NS3 protease, by targeting a host factor upstream of IRF-3 (22). Many other RNA and DNA viruses have proteins with demonstrated antagonism for the IFN-/ system. In the case of BVDV, suppression of IFN-/ production has traditionally been described as a fundamental property of ncp biotype strains, whereas its role in cp BVDV infections was incompletely defined (6, 12, 66). The similar levels of NS2-3 expression in cells infected with N-dINS and N-INS-L8P suggest that NS2-3 expression may not be essential to prevent IFN-/ induction.
These findings also have implications for pestivirus vaccine design. The manipulation of the N-terminal region of Npro may allow the engineering of avirulent viruses which could be used as live vaccines. Because suppression of innate immune responses is likely to prove critical for the establishment of persistent fetal infections, it is tempting to speculate that these viruses may be safer than existing vaccines for the developing fetus.
The identification of Npro as a suppressor of IFN-/ is also consistent with a significant amount of experimental and circumstantial data that associate BVDV infections with immunosuppression. In vitro studies have shown that lymphocytes, macrophages, and neutrophils from normal donor animals infected with BVDV were functionally impaired (37, 52, 62). BVDV infections are known to increase susceptibility to other pathogenic bacteria (58, 59) and viruses, e.g., BVDV and respiratory syncytial virus coinfections result in enhanced respiratory syncytial virus lung pathology (10). The immunosuppression caused by BVDV infection also interferes with immunological assays for Mycobacterium bovis, which compromises the diagnosis of tuberculosis (13). Further studies are needed to determine if these interactions involve the suppression of IFN-/ by Npro.
ACKNOWLEDGMENTS
This work was supported by USDA/NRI grant 2002-35204-11619 to R.O.D.
We are grateful to all the current and past members of the Donis laboratory for reagents, protocols, and helpful discussions. We also thank F. Osorio for generous gifts of virus strains. We are indebted to L. Babiuk for a gift of IFN-, to L. Zhang for a gift of pIFN--CAT, and to X. Cao for antibodies.
Present address: Viral Vaccines Research and Development, GlaxoSmithKline Biologicals, Rue de l'Institut, 89 (P31-005), B-1330 Rixensart, Belgium.
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Population and Diagnostic Medicine, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 18453
Valeant Pharmaceuticals, Valeant Plaza, 3300 Hyland Avenue, Costa Mesa, California 92626
ABSTRACT
The alpha/beta interferon (IFN-/) system is the first line of defense against viral infection and a critical link between the innate and adaptive immune responses. IFN-/ secretion is the hallmark of cellular responses to acute RNA virus infections. As part of their survival strategy, many viruses have evolved mechanisms to counteract the host IFN-/ response. Bovine viral diarrhea virus (BVDV) (genus Pestivirus) was reported to trigger interferon production in infected cultured cells under certain circumstances or to suppress it under others. Our studies with various cultured fibroblasts and epithelial bovine cells indicated that cytopathic (cp) BVDV induces IFN-/ very inefficiently. Using a set of engineered cp BVDVs expressing mutant Npro and appropriate controls, we found that the IFN-/ response to infection was dependent on Npro expression and independent of viral replication efficiency. In order to investigate whether the protease activity of Npro is required for IFN-/ antagonism, we engineered Npro mutants lacking protease activity by replacement of amino acid E22, H49, or C69. We found that E22 and H49 substitutions abolished the ability of Npro to suppress IFN, whereas C69 had no effect, suggesting that the structural integrity of the N terminus of Npro was more important than its catalytic activity for IFN-/ suppression. A catalytically active mutant with a change at a conserved Npro region near the N terminus (L8P) in both BVDV biotypes did not antagonize IFN-/ production, confirming its involvement in this process. Taken together, these results not only provide direct evidence for the role of Npro in blocking IFN-/ induction, but also implicate the amino-terminal domain of the protein in this function.
INTRODUCTION
The genus Pestivirus comprises an important group of animal pathogens that includes agents such as Bovine viral diarrhea virus (BVDV) and Classical swine fever virus (CSFV). Based on their molecular phylogenies, as well as physical, chemical, and biological properties, the genus Pestivirus is classified in the family Flaviviridae (74). The pestivirus genome consists of a single RNA molecule approximately 12 kb in length and with positive polarity (14, 43). The enveloped virions deliver the genomic RNA to target cells by fusion with endocytic membranes (21). Pestiviruses replicate exclusively in the cytoplasm, generally without inducing death of the infected cultured cells, i.e., most pestiviruses are noncytopathic (ncp) (27).
Much of the economic impact of pestivirus infections results from their tropism for the fetus. Fetal infection with ncp BVDV during the first trimester of gestation results in the birth of calves that are persistently infected and immunotolerant to the viral antigens. These newborn calves develop into apparently healthy animals, which remain viremic for life. The ncp BVDV virus pool within persistently infected animals often gives rise to cytopathic (cp) mutants, which cause a fatal syndrome known as mucosal disease (8, 11, 46, 47, 49, 54). Cp BVDV variants induce apoptosis in cultured cells (29, 30, 32, 76, 81). It is thought that immunologic tolerance allows unchecked replication of these cp BVDV variants, triggering the pathogenic cascade associated with mucosal disease (9). The biochemical hallmark of cp variants is the production of high levels of NS3 as a free protein after the early phase of infection (17, 39, 57). In contrast, ncp BVDV variants produce largely NS2-3 and small amounts of NS3 during the early phase of infection (39). The pathogenesis of the fatal mucosal disease syndrome is poorly characterized; however, excessive stimulation of innate immune responses may play a significant role. Alpha/beta interferons (IFN-/) are powerful cytokines which regulate cells from most organ systems in the body with regard to their permissiveness to viral infection and many other specialized functions, e.g., differentiation of dendritic cells (25, 50, 55, 70).
The production of IFN-/ in virus-infected cells is transcriptionally regulated (45). IFN-/ gene transcription is activated by multiple signals, the best characterized being viral infection, in which signaling is thought to actually be mediated by double-stranded RNA (dsRNA) (26, 38, 79). Viral infections, particularly those caused by RNA viruses, involve the production of cRNA molecules in the form of extended dsRNA structures. dsRNA, as well as endoplasmic reticulum stress, oxidative stress, and additional uncharacterized viral signals, activates a cascade of events that converge to activate several transcription factors, such as NF-B and ATF-2/c-jun, as well as interferon regulatory factor 3 (IRF-3) and IRF-7 (2, 4, 35, 38, 68, 69, 79). The resulting enhanceosome is responsible for a surge of IFN-/ gene transcription, leading to translation and secretion. IFN-/ molecules bind interferon receptors (IFN-/R) present in virtually all nucleated cells of the body to activate an antiviral program in the target cells. To counter this antiviral response of host cells, many viruses have evolved effectors that actively interfere with the IFN-/ system or with the downstream interferon-stimulated gene products that mediate the antiviral state (7, 23, 24, 28, 48, 51, 73, 78, 82). Viruses that target the cellular IFN-/ system have two general modes of action: they either suppress the production of IFN-/ or interfere with the IFN-/R signaling pathway (7, 24, 67). Previous studies have shown that infection of cultured cells with cp BVDV triggers IFN-/ production in macrophages and in some nonmyeloid tissue cultures, whereas no IFN-/ is produced by ncp-BVDV-infected cells of any type (1, 6, 56, 66). However, both cp and ncp BVDVs have been shown to block the function of IRF-3, which is required for IFN-/ transcription (5, 6). Thus, cp-BVDV infection is reported to either induce or suppress IFN-/ in infected cells. The Npro and Erns proteins have been reported to participate in IFN-/ suppression during CSFV infection (34, 41, 64). The Npro protein is dispensable for the growth of BVDV and CSFV in cell culture, but deletion mutants are growth impaired in cell culture (40, 71). The growth impairment of CSFV Npro deletion mutants has been recently linked to interferon antagonism (64). Recently, it was demonstrated that the Npro protein of CSFV can impair IRF-3 activity (33, 41). The genetic analysis of Npro mutants of pestiviruses is complicated by the fact that changes in the RNA sequence that encode this protein can affect polyprotein translation and cleavage of the capsid protein, as well as host cell IFN-/ responses. The RNA sequences encoding Npro that are adjacent to the internal ribosome entry site (IRES) element modulate translation of the viral polyprotein (53, 60). In this report, we show that cp-BVDV infections do not induce detectable levels of IFN-/ and that the phenotype depends on Npro expression. We also found that antagonism to IFN-/ production by viral infection requires the N-terminal domain of Npro, whereas its endoproteinase function seems dispensable for this purpose.
MATERIALS AND METHODS
Cells and viruses. Bovine testicle cells express epithelial markers and were derived as described previously (18). NCL1 bovine uterine cells are also epithelioid (E. J. Dubovi, unpublished data), whereas CRIB cells resistant to BVDV infection (20) were derived from MDBK cells. All cells were grown in minimum essential medium (MEM) supplemented with 10% equine serum (ES; HyClone) and 1x penicillin-streptomycin antibiotics (Life Technologies). The NCL1-ISRE-Luc-Hygro cell line (L. Gil and R. Donis, unpublished data) is a stable bovine cell line selected for integration of the luciferase reporter under transcriptional control of an IFN-stimulated response element (ISRE); it was cultured in the same medium supplemented with 300 μg/ml hygromycin (A. G. Scientific, Inc.).
pNADLp15 (cp NADL), N-dINS (ncp NADL), N-H/B, and 5B-1HR are modified viruses derived from a plasmid containing the full-length cDNA of the BVDV strain NADL genome, as described elsewhere (3, 40, 76). The mutant 5B-1HR was derived by reverse genetics from the NADL strain of BVDV by insertion of a 19-amino-acid epitope tag at position 704 of NS5B (insertion of 57 nucleotides at position 12303) (3). Vesicular stomatitis virus (VSV) New Jersey serotype was obtained from F. Osorio (University of Nebraska).
Antibodies. BVDV Npro polyclonal antibody (kindly provided by X. Cao, YC Inc., Boston, MA), NS3 monoclonal antibody 20.10.6 (15), horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) antibody (Sigma) and HRP-conjugated anti-mouse IgG antibody (Sigma) were used for Western blot analysis. The goat anti-BVDV polyvalent antiserum (VMRL Inc.) and secondary recombinant HRP-conjugated protein G (Zymed) were used for focus assays.
Virus preparation. All experiments aimed at measuring IFN-/ responses to virus infection were performed using pelleted virus preparations to infect cells. This step removed IFN-/ and other cytokines that could be present in the cell culture fluids harvested to prepare BVDV stocks. Virus stocks were produced by inoculating NCL1 cells with seed virus at an input multiplicity of infection (MOI) of 1. The cell culture fluid was harvested at 24 h postinfection (p.i.) and clarified by low-speed centrifugation (2,000 x g) for 30 min. The clarified culture fluid was centrifuged at 100,000 x g for 2 h. The supernatant was discarded, and the virus-containing pellet was drained, resuspended in a small volume of MEM with 5% horse serum, and frozen in aliquots. Stocks used in these studies had titers of 7.5 to 7.7 log10 median tissue culture infective doses/ml (36).
Virus infection. Bovine cells were infected with pelleted virus stocks at an MOI of 10 in MEM containing 5% equine serum. After an absorption period of 1 h, the inoculum was removed and replaced with fresh medium.
Virus growth kinetics. To determine the viral replication efficiency, bovine uterine cells were infected with each virus at an MOI of 0.3. After 1 h of incubation at 37°C, the inoculum was removed and the cell monolayers were washed three times with MEM. Fresh MEM containing 5% ES was added, and the cells were incubated at 37°C. The supernatants were harvested at different times after infection, and their infectious titers were determined by serial dilution.
Plaque assay. Bovine testicle cell monolayers were infected with serial 10-fold dilutions of the viruses. Following 1 h of adsorption at 37°C, the inoculum was removed, and the cells were overlaid with 0.8% agarose in MEM plus 5% equine serum and incubated at 37°C. After 4 days, the cells were fixed and stained with 0.05% crystal violet in 10% formalin.
Focus assay. The plaque assay was done as indicated above. After 4 days, the cells were fixed with cold 30% acetone in phosphate-buffered saline (PBS) and air dried. The monolayers were sequentially incubated for 90 min with goat anti-BVDV polyvalent antiserum, followed by a 60-min incubation with recombinant HRP-conjugated protein G. Both were diluted in PBS containing 0.01% Tween 20 and 0.5 M NaCl. Bound conjugates were revealed with the peroxidase substrate 3-amino-9-ethylcarbazole (Sigma).
Construction of BVDV mutants and sequence analysis. BVDV mutants were generated using a reverse genetics system established previously (42, 75). The introduction of mutations was confirmed by reverse transcription (RT)-PCR and sequence analysis. Descriptions of constructs are presented below. Viral RNA was extracted using a commercial lysis solution and resin kit (RNAeasy; QIAGEN). RT-PCR was carried out using a one-step RT-PCR kit (QIAGEN). cDNA products were purified using QIAquick PCR purification (QIAGEN). Sequencing reactions were performed using the ABI BigDye terminator cycle-sequencing kit with products resolved on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The sequences of primers used for amplification and sequencing are available upon request. DNA sequence analysis was performed using version 10 of the Genomics Computer Group sequence analysis package (16).
RNA transcription and electroporation. RNA transcripts were prepared using a MEGAscript in vitro transcription kit as recommended by the manufacturer (Ambion). The concentration of purified RNA was determined by staining with RiboGreen RNA quantification reagent (Molecular Probes), as described previously (77).
The in vitro-transcribed RNAs were transfected into bovine uterine cells by electroporation as described previously (3). Briefly, bovine uterine cells were trypsinized, washed two times with MEM, and resuspended at 2 x 107 cells/ml in cytomix (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4, pH 7.6, 25 mM HEPES, pH 7.6, 2.0 mM EGTA, 5.0 mM MgCl2) plus 2.0 mM of ATP and 5.0 mM of glutathione (72). Unless otherwise indicated, 3 μg of transcribed RNA was mixed with 0.1 ml of cells in suspension and immediately pulsed with a BTX-600 ElectroSquarePorator (320 V; 100 μF; 48 ). The electroporated cells were diluted in 10 ml of MEM-ES and plated on tissue culture dishes.
RNA infectivity determination by infectious-center assay. An infectious-center assay was used to quantify the specific infectivity of in vitro-transcribed RNA. Tenfold dilutions of electroporated bovine uterine cells were added to preseeded monolayers of MDBK cells grown to 60% confluence. Following 4 h of incubation, the cells were washed and overlaid with 0.8% agarose in MEM-5% equine serum. The plates were incubated for 3 days at 37°C, and infectious centers were visualized and counted by staining the plaques as described above.
Northern blots. Bovine testicle cells (106 cells; 60-mm dish) were infected with purified BVDV at 10 PFU/cell. At 1 h postinfection, the cell monolayers were washed with 5 ml of PBS, followed by the addition of 5 ml of MEM containing 5% ES. The total RNA from bovine uterine cells was prepared by using an RNeasy total-RNA kit (QIAGEN). Five micrograms of total RNA was separated in a formaldehyde-containing agarose gel, transferred onto a nylon membrane (Hybond N+; Amersham Biosciences), and probed by hybridization to a DNA fragment spanning nucleotides 6044 to 6432 of BVDV-NADL labeled with [-32P]dATP by a random priming method (Prime-a-Gene Labeling System, Promega). The membranes were washed twice in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS) at room temperature and once in 0.1x SSC-0.1% SDS at 60°C for 30 min and exposed to film (X-Omat; Kodak). The blots were rehybridized with GAPDH (glyceraldehyde phosphate dehydrogenase) probe to ascertain the lane loading and transfer efficiency. Scanning densitometry was achieved by PhosphorImager analysis with ImageQuant software (Molecular Dynamics). The signal intensities from BVDV-specific bands were normalized by comparison to the signal from the cellular housekeeping gene GAPDH mRNA.
Western blots. Proteins were separated by SDS-polyacrylamide gel electrophoresis (10% or 12% acrylamide) and transferred onto a nitrocellulose membrane (HybondTM; Amersham Life); the membrane was blocked with 5% (wt/vol) dried skim milk in PBS containing 0.005% Tween 20. For the detection of the Npro protein, membranes were probed with polyclonal antibody specific against the Npro protein of BVDV, followed by incubation with a secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody. The NS3 or NS2-3 protein of BVDV was detected by using the monoclonal antibody 20.10.6, which is specific for the pestivirus nonstructural protein NS3, and the bound antibodies were detected by horseradish peroxidase-conjugated anti-mouse IgG antibody (15). The membranes were rinsed five times in PBS containing 0.005% Tween 20 after primary and secondary antibodies. The membrane-bound immune complexes were revealed by the enhanced-chemiluminescence detection system (Amersham), and images were captured on X-ray film.
Reporter plasmids and electroporation. pIFN-beta-CAT expresses choramphenicol acetyltransferase (CAT) under the control of the IFN- enhancer element (19), pISRE-Luc expresses firefly luciferase under the control of an interferon-stimulated response element (Stratagene) (31), and pRL-SV-40 (Promega) expresses Renilla luciferase under the transcriptional control of the simian virus 40 promoter element. Reporter plasmids were delivered into cells by electroporation of DNA into cells in suspension. Briefly, bovine uterine cells (90% confluent) were trypsinized, washed twice with MEM, and resuspended at 2 x 107 cells/ml in cytomix (72). Unless otherwise indicated, 3 μg of reporter DNA plasmid was mixed with 0.1 ml of cells in suspension and immediately pulsed with a BTX-600 ElectroSquarePorator (320 V; 100 μF; R3) as previously described (3). The electroporated cells were diluted in 12 ml of MEM-ES and seeded (1 ml per well) into 12-well plates. The plates were incubated for 4 h at 37°C, after which the medium was replaced with fresh medium. At 24 h postelectroporation, the cells were mock infected or infected with BVDV at an MOI of 10. At the indicated times after infection, cells were lysed in reporter lysis buffer (Promega). CAT assays were performed using 14C-labeled chloramphenicol and n-butyryl coenzyme A, following the CAT assay kit instructions (Promega). The luciferase assay was performed by using the dual-luciferase (firefly and Renilla) assay system (Promega) according to the manufacturer's protocol. Expression levels were normalized for transfection efficiency by dividing the firefly luciferase or CAT values by those of Renilla luciferase. The relative expression of reporter enzyme (change [n-fold]) was calculated by dividing each test sample into the mock-infected control value.
IFN-/ bioassay. The production of IFN-/ in BVDV-infected cells was determined according to the method of Rubinstein et al. (63) with some modifications. Bovine cells were infected with purified BVDV or BVDV mutants at an MOI of 10. At 24 h postinfection, the medium was clarified by centrifugation. In order to inactivate BVDV and other cytokines present in the samples, cell culture supernatants were adjusted to pH.2 with a 2 M hydrochloric acid solution. After 24 h at 4°C, the pH was restored to 7 with a 2 M sodium hydroxide solution. Serial dilutions in MEM-5% ES were added to 96-well plates and overlaid with 0.1 ml of MEM-5% ES containing CRIB cells at a concentration of 3 x 105 cells per ml. After incubation in a moist chamber at 37°C for 16 h, CRIB cell monolayers were infected with VSV at an MOI of 0.1. After incubation for an additional period of 24 h, the monolayers were fixed and stained with crystal violet and the IFN titer was determined as follows. The number of IFN equivalent units in the experimental samples that conferred 50% protection from the cytopathic effect of VSV was determined by comparison with samples analyzed in parallel using titrated units of recombinant bovine IFN- as a standard (kindly provided by Lorne Babiuk, VIDO, Saskatchewan, Canada).
IFN reporter gene assay. The IFN-/ activity was also measured by using the NCL-1-ISRE-Luc-Hygro cells, a stable bovine cell line with the ISRE-Luc reporter cassette integrated into its genome (Gil and Donis, unpublished). Briefly, 1.5 x 105 cells were seeded into a 12-well plate and cultured at 37°C. After incubation in a moist chamber at 37°C for 12 h, the medium was replaced with 0.5 ml of test sample and incubated for 8 h. Cells were harvested and lysed in 100 μl of passive lysis buffer (Promega), and the firefly luciferase activity was measured as described above. This assay is sensitive to bovine IFN-/ and detected IFN-/ levels as low as 5 IU/ml (Gil and Donis, unpublished).
PINBA. Bovine testicle cell were infected with ncp BVDV (MOI = 1) and passed twice before being used for the poly(I · C) for ncp BVDV assay (PINBA). The PINBA was performed as described previously (44, 61) with some modification. Bovine testicle cells, uninfected or persistently infected with ncp BVDV, were seeded into a 96-well plate at a concentration of 3 x 104 cells/well and incubated for 6 h at 37°C. After incubation, the medium was removed and 50 μl of fresh medium per well with or without 50 μg of poly(I · C) per ml was added. Twelve hours later, the cell monolayers were challenged with VSV infection at an MOI of 0.1. Following overnight incubation, the monolayers were stained with crystal violet to better visualize the VSV cytopathic effects.
RESULTS
IFN-/ secretion by cells infected with Npro mutants of BVDV. The presence of a nonstructural protein at the 5' end of the polyprotein is a defining feature of pestiviruses. This polypeptide, termed Npro, is an unconventional protease capable of autocatalytic cleavage at its own C terminus (65, 80). To characterize the role of the Npro protein, a series of Npro BVDV mutants were used (Fig. 1). Two of the Npro mutants were deletion mutants, one (N-dNpro) in which the Npro protein was completely deleted and the other (N-H/B) in which Npro was replaced with a heterologous autocatalytic cleavage cassette derived from the hepatitis C virus (HCV) NS3 protease domain. The two Npro deletion mutants were previously found to be phenotypically similar (40). The growth curve of a mutant virus lacking Npro showed that the virus progeny levels were 10-fold lower than those of wild-type (wt) virus at each of the time points (Fig. 2B) (40). This observation was correlated with an 3-fold reduction of specific infectivity of the RNA compared to that of the wt virus (Fig. 2A). The infectivity of the RNA, growth kinetics, plaque size, and RNA accumulation revealed that the N-H/B chimeric virus is similar to the N-dNpro mutant in its replication efficiency (Fig. 2A to D). The third Npro mutant, termed N-Npro18GFP, contains the jellyfish green fluorescent protein (GFP) gene cloned within the N-terminal region of Npro, between codons 18 and 19 (Fig. 1). Although the autocatalytic protease activity of this Npro chimera remained intact (data not shown), the virus formed small plaques like the other two mutants (Fig. 2C). The analysis of cellular responses to infection by Npro deletion and chimeric mutants is complicated by the fact that Npro mutant viruses have reduced replication efficiency and, consequently, delayed induction of apoptosis compared to the wt virus. Thus, to test the hypothesis that increased cytokine production and secretion in cells infected by Npro mutants is not attributable to a decreased impairment of host cell metabolism compared to that of cells infected by wt virus, we also developed and characterized an isogenic BVDV mutant with an altered NS5B polymerase gene, termed 5B-1HR. Comparative analysis of this mutant with the Npro deletion viruses revealed that it displays a degree of replication deficiency similar to that observed with Npro mutants (Fig. 2C and D).
We used our panel of mutant chimeric and mutant cp BVDVs and several bovine cell lines to assess the effects of viral infection on IFN-/ production. To this end, we first used the VSV bioassay to measure IFN-/ levels in the culture media of infected cells. No detectable IFN was present in the culture media of cells infected with wt NADL or the ncp derivative N-dINS, whereas media from cells infected with Npro mutants showed high levels of IFN-/ (Table 1). Although the levels of IFN-/ varied among cell lines, three different cell lines with diverse origins—uterine, testicular, and renal—showed the same pattern of response to infection, suggesting that the consequence of viral infection was reproducible (Table 1). Bovine cells lacking BVDV receptor function (CRIB) inoculated with Npro mutant viruses showed no detectable IFN-/ in the medium, suggesting that induction of IFN-/ production was dependent on viral infection and did not involve signaling through plasma membrane receptors (20). CRIB cells were otherwise responsive to another IFN-/ inducer, poly(I · C) (Table 1).
We used the 5B-1HR linker insertion mutant, which is phenotypically very similar to the mutants with Npro deleted with regard to plaque size and viral RNA accumulation, to determine whether the cellular IFN-/ response was a correlated with viral replication efficiency or Npro expression (Fig. 2C and D). We found that mutant 5B-1HR-infected cells responded like the wt-virus-infected cells: no detectable levels of IFN-/ were present in the culture medium (Table 1). Thus, high levels of IFN-/ secretion after infection with growth-impaired mutants are correlated with a lack of intact Npro expression and not with reduced cytopathogenicity for the infected cells.
IFN-/ and ISRE gene transcription in BVDV-infected cells. The high levels of IFN-/ released into the media of cells infected with Npro deletion mutants could result from transcriptional or posttranscriptional regulation events. To investigate these possibilities, cells were transiently transfected with human IFN--CAT reporter or ISRE-Luc reporter plasmids and subsequently infected with wt or Npro mutant viruses. Cells infected with the Npro mutants showed 40- to 70-fold- and 80- to 145-fold-increased levels of activation of the IFN- and ISRE promoters at 20 h p.i. compared to the wt virus or the 5B-1HR mutant (Fig. 3A and B). Cells infected with N-dINS (ncp NADL) and wt NADL showed background levels of IFN- promoter activation identical to those of mock-infected cells, in agreement with our earlier data (Table 1). These results implicate IFN-/ gene transcription regulation as the major determinant of secreted cytokine levels and reveal the possible autocrine or paracrine action of the secreted IFN-/ in the activation of the ISRE enhancer element.
Role of the catalytic domain of Npro in IFN-/ antagonism. Recent studies showed that the HCV NS3/4A serine protease blocks the phosphorylation of IRF-3, a key cellular antiviral signaling molecule (22). To investigate the importance of the proteinase activity of Npro in IFN-/ antagonism, we utilized a reverse genetics approach targeting residues E22, H49, and C69, individually known to be essential for the catalytic activity of Npro in pestiviruses (65). However, mutations that abolish the proteinase activity of Npro are lethal due to improper maturation of the capsid protein, which in turn prevents the formation of infectious virions (40). To overcome this problem, we engineered a virus whose genome consisted of the N-H/B genome (Fig. 1) with the Npro protein cloned upstream of HCV NS3 protease (Fig. 4A). The restored virus, termed N-Npro-H/B, produced plaques slightly larger than those of the parental virus N-H/B (2.45 ± 0.44 mm in diameter versus 1.7 ± 0.29 mm), which we hypothesized stemmed from the restoration of Npro function (Fig. 4B). To determine whether the improved growth phenotype of N-Npro-H/B involved IFN-/ inhibition, we analyzed the production of IFN-/. No detectable IFN-/ activity was present in the medium of bovine cells infected with N-Npro-H/B, confirming previous observations that the Npro protein has IFN-/ antagonist properties (Fig. 4C). Most importantly, the endoproteolytic activity of Npro in this genome is predicted to be dispensable for release of the capsid protein C. Therefore, the genome was deemed suitable for engineering Npro catalytic mutants. Mutants of residues previously described as critical for the protease activity of Npro were engineered in the N-Npro-H/B infectious clone, giving rise to the following viable viruses from full-length genomes: N-Npro-H/B-E22L, N-Npro-H/B-H49V, and N-Npro-H/B-C69A (Fig. 5A). The consequences of these mutations for Npro endoproteinase activity were determined by tracing Npro in infected cell lysates by Western blotting with a specific polyclonal antibody (Fig. 5B). Npro expression was readily detected as a 20-kDa band in blots from lysates of cells infected with wt and N-Npro-H/B. In contrast, the three protease mutant viruses lacked the 20-kDa protein and expressed only a fusion protein of approximately 40 kDa, comprising the mutated Npro and the HCV protease. Interestingly, the cleavage of Npro by N-Npro-H/B is incomplete, and substantial amounts of the Npro-HCV protease fusion protein are detected. Regardless of the cause of the reduced catalytic function of Npro as an endoproteinase, the plaques formed by the parental virus N-NproH/B and the N-NproH/B-C69A mutant were comparable in size (1.3 ± 0.3 mm and 1.4 ± 0.3 mm in diameter, respectively), suggesting similar replication efficiencies. However, the plaques produced by the N- NproH/B-E22L and N- NproH/B-H49V mutants were smaller (0.6 ± 0.19 mm and 0.76 ± 0.29 mm, respectively) (Fig. 5C).
We next analyzed IFN-/ induction in the culture media of cells infected with each protease mutant (Fig. 5D). Interestingly, cells infected with the E22 and H49 mutants induced high levels of IFN. In contrast, the C69 mutant induced background levels of IFN-/, suggesting that the catalytic endoproteinase activity of Npro is dispensable for the IFN-/ antagonist function. In this context, the lack of IFN-/ antagonism of mutants E22 and H49 could be explained by disruption of a hypothetical IFN-/ regulatory domain located near the N terminus of Npro.
A conserved domain at the N terminus of Npro is essential for IFN antagonism. Alignment of multiple pestivirus Npro sequences revealed a 5-amino-acid sequence motif (LLYKT; codons 8 to 12) near the amino terminus of the protein as the largest conserved block in the region. In order to study the contribution of this conserved domain to IFN antagonism, we constructed a mutant viral genome termed N-L8P, in which the highly conserved leucine at position 8 was changed to proline. The N-L8P virus formed considerably smaller plaques than the wt virus (1.9 ± 0.4 mm versus 2.9 ± 0.95 mm in diameter), resembling the plaques formed by the viruses with Npro deleted (Fig. 6A). This was correlated with an 68% reduction in progeny yield at various times after infection (Fig. 6D). The stabilities of Npro and its endoproteinase were examined by Western blot analysis of the steady-state levels of the protein in infected cells. The Western blots of bovine testicle cells infected with the N-L8P mutant revealed that Npro expression levels and endoproteinase activity were similar to those of the parental viruses (Fig. 6B). Interestingly, culture medium from cells infected with N-L8P contained high levels of IFN-/, as indicated by a 60-fold increase in luciferase expression in reporter cells relative to controls (Fig. 6C). Thus, the impaired growth of N-L8P was correlated with the induction of IFN-/ production in the infected cells. Similarly high levels of IFN-/ were detectable in the medium from cells infected with N-dNpro, which yielded a 56-fold increase of reporter gene expression. These data indicated that a single amino acid change (L8P) within the N terminus of Npro resulted in a virus that is phenotypically identical to the Npro deletion mutants with regard to IFN-/ induction.
To increase our understanding of the IFN-/ antagonistic function of the BVDV Npro protein, we create the L8P ncp mutant virus. Figure 7A shows the focus-forming phenotypes of N-dINS (the ncp NADL parental virus) and N-dINS-L8P (the ncp L8P mutant virus); both viruses displayed focus formation with similar sizes (2.3 ± 0.7 mm versus 2.2 ± 0.5 mm in diameter, respectively). Levels of NS2-3 expression were examined by Western blot analysis of cell lysates infected at an MOI of 5 PFU/cell and collected at 18 h p.i. (Fig. 7B). Unsurprisingly, the levels of NS2-3 and NS3 expression were higher for the wt NADL than N-L8P. However, uncleaved NS2-3 levels were similar for N-dINS and N-dINS-L8P. IFN-/ analysis (Fig. 7C) using an ISRE luciferase reporter cell line indicated that supernatants from mock-infected cells or cells infected with wt NADL and N-dINS did not contain IFN-/ activity. Interestingly, supernatants from cells infected with N-dINS-L8P showed a very low level of IFN, which resulted in a 1.5-fold increase in luciferase expression. In contrast, supernatant from N-L8P-infected cells induced a 58-fold increase in luciferase expression. These results suggest that N-dINS-L8P is a poor IFN inducer in cultured cells, and this characteristic might be due to the low levels of viral-RNA accumulation present in ncp-BVDV-infected cells (76).
Next, we tested whether N-dINS-L8P virus could block IFN-/ induction by dsRNA treatment. For this purpose, we used the PINBA previously described (44, 61). The results of this experiment demonstrated that, unlike N-dINS, cells persistently infected with N-dINS-L8P virus are not able to inhibit IFN-/ induction by dsRNA and consequently were nonsusceptible to VSV infection (Table 2). Taken together, these results strongly suggest that the N-terminal domain of the BVDV Npro protein plays a critical role in IFN-/ suppression.
DISCUSSION
We exploited a reverse genetics approach to analyze the role of Npro in virus-host interactions. We first studied two BVDV mutants with Npro deleted and one Npro mutant derived from the same infectious clone but structurally distinct at the junction between the IRES element and the polyprotein open reading frame. We also analyzed a mutant with intact Npro that is phenotypically similar to the mutants with Npro deleted with regard to replication efficiency and RNA accumulation. Contrary to the prevailing notion that wt NADL infection induced IFN-/, we found that, at least in our systems, infection with wt NADL was indistinguishable from mock infection or ncp BVDV infection. Assay sensitivity cannot explain these differences, because we used a highly sensitive reporter cell line to detect IFN-/ in concentrations as low as 5 IU/ml (data not shown). Because these observations were the same in different cell lines, we cannot attribute the failure of wt NADL to induce IFN-/ to host factor variability. Analysis of IFN-/ in the supernatants of bovine cells infected with different cp BVDV strains detected only low levels of IFN-/, which suggests that cp BVDV is a poor IFN-/ inducer (data not shown).
In contrast to the wt NADL, mutants with Npro deleted elicited very high levels of IFN-/, regardless of the type of bovine cell line used. The unrestrained IFN-/ production triggered by infection with an Npro deletion mutant appeared to contribute significantly to the reduced growth of these mutants. The reduced efficiency of IRES function in the Npro deletion mutants might further contribute to the replicative impairment.
The importance of Npro for IFN-/ suppression was made clear by the restoration of Npro to the N-H/B deletion mutant virus. N-Npro-H/B showed no IFN-/ induction and produced plaques larger than those of the parental virus (N-H/B), which induced IFN-/ production. Interestingly, transient expression of Npro by plasmid transfection did not result in functional complementation; the growth of the Npro deletion mutants could not be rescued, presumably because of inefficient Npro expression in transfected bovine cells. Previous reports implicating a viral proteolytic activity in the suppression of host IFN-/ response by HCV NS3 prompted us to examine whether the Npro endoproteinase has an analogous function (22). Mutagenesis of Npro effectively disabled its catalytic activity, confirming the findings of Rumenapf et al. (65). One of the catalytically inactive Npro mutants (C69) showed efficient suppression of IFN-/ induction, strongly suggesting that the catalytic activity of Npro is dispensable for this function, although the unlikely possibility that an undetected residual protease activity is responsible for IFN-/ suppression cannot be ruled out. However, the two other mutants (E22 and H49) with a catalytically disabled Npro lost IFN-/-antagonistic activity. Although this could point to the importance of the catalytic activity of Npro for IFN-/ suppression, it is also possible that the structural changes that disable catalysis also disrupt an overlapping domain involved in IFN-/ antagonism. The second hypothesis is consistent with the identification of L8 as a critical amino acid in a conserved region of the N-terminal domain. Lack of IFN-/ suppression by mutants L8P, E22L, and H49V suggests that the N-terminal 49 amino acids of Npro are essential for the suppression of IFN-/ production in infected cells. In addition, the cloning of N-dINS-L8P is consistent with the IFN-/-antagonistic properties of the N-terminal domain of the Npro protein and demonstrated that the L8 mutation in the ncp NADL strain abolished the ability of the ncp virus to inhibit IFN-/ induction by dsRNA treatment. Surprisingly, cells infected with N-dINS-L8P did not induced high levels of IFN-/ production as expected. One reason for this unexpected outcome of N-L8P versus N-dINS-L8P might be related to different amounts of dsRNA being formed during replication. Previous data showed that the amount of RNA present in cells infected with cp BVDV vastly exceeds that in cells infected with ncp biotypes (76). Therefore, it is tempting to speculate that a large amount of this viral RNA is implicated in the IFN-/-inducing activity of the N-L8P virus.
The identification of Npro as a potential suppressor of IFN-/ production by BVDV-infected cells provides a candidate target for intervention, since it is becoming increasingly clear that this function is part of the essential machinery of animal viruses. HCV was recently shown to have developed a mechanism to suppress IFN-/ transcription activation by way of its NS3 protease, by targeting a host factor upstream of IRF-3 (22). Many other RNA and DNA viruses have proteins with demonstrated antagonism for the IFN-/ system. In the case of BVDV, suppression of IFN-/ production has traditionally been described as a fundamental property of ncp biotype strains, whereas its role in cp BVDV infections was incompletely defined (6, 12, 66). The similar levels of NS2-3 expression in cells infected with N-dINS and N-INS-L8P suggest that NS2-3 expression may not be essential to prevent IFN-/ induction.
These findings also have implications for pestivirus vaccine design. The manipulation of the N-terminal region of Npro may allow the engineering of avirulent viruses which could be used as live vaccines. Because suppression of innate immune responses is likely to prove critical for the establishment of persistent fetal infections, it is tempting to speculate that these viruses may be safer than existing vaccines for the developing fetus.
The identification of Npro as a suppressor of IFN-/ is also consistent with a significant amount of experimental and circumstantial data that associate BVDV infections with immunosuppression. In vitro studies have shown that lymphocytes, macrophages, and neutrophils from normal donor animals infected with BVDV were functionally impaired (37, 52, 62). BVDV infections are known to increase susceptibility to other pathogenic bacteria (58, 59) and viruses, e.g., BVDV and respiratory syncytial virus coinfections result in enhanced respiratory syncytial virus lung pathology (10). The immunosuppression caused by BVDV infection also interferes with immunological assays for Mycobacterium bovis, which compromises the diagnosis of tuberculosis (13). Further studies are needed to determine if these interactions involve the suppression of IFN-/ by Npro.
ACKNOWLEDGMENTS
This work was supported by USDA/NRI grant 2002-35204-11619 to R.O.D.
We are grateful to all the current and past members of the Donis laboratory for reagents, protocols, and helpful discussions. We also thank F. Osorio for generous gifts of virus strains. We are indebted to L. Babiuk for a gift of IFN-, to L. Zhang for a gift of pIFN--CAT, and to X. Cao for antibodies.
Present address: Viral Vaccines Research and Development, GlaxoSmithKline Biologicals, Rue de l'Institut, 89 (P31-005), B-1330 Rixensart, Belgium.
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