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Foot-and-mouth disease virus (FMDV) is a positive-stranded RNA virus capable of infecting a variety of domestic and wild biungulate species (Pega et al., 2013). The highly contagious foot-and-mouth disease (FMD) is caused by FMDV and is perhaps the most important limiting factor in the trade of animals and animal products (Barasa et al., 2008; Perry et al., 2007; Rufael et al., 2008), with outbreaks usually resulting in large economic losses for the local livestock industry. FMDV belongs to the Aphthovirus genus of the Picornaviridae family, and its genome is a single-stranded, positive-sense RNA that encodes a polyprotein. The polyprotein is post-translationally cleaved by three viral proteinases, leader (Lpro), 2A, and 3Cpro, into precursors and mature viral structural (VP4, VP2, VP3, and VP1) and nonstructural proteins (Lpro, 2A, 2B, 2C, 3A, 3B, 3Cpro, and 3D) (Racaniello, 2007).
Translation of the FMDV polyprotein begins at two different AUG start codons separated by 84 nucleotides, resulting in two alternative forms of Lpro designated as Labpro and Lbpro (Clarke et al., 1985; Piccone et al., 1995). During its evolution, FMDV has consistently counteracted against host immune systems to facilitate its survival and replication; several mechanisms have evolved to antagonize host immune responses, with Lpro reported to play significant pathogenic roles (Grubman et al., 2008). Lpro is a well-characterized, papain-like proteinase (Medina et al., 1993; Piccone et al., 1995) that can self-cleave from the nascent polyprotein. Host translation-initiation factor eIF-4G can also be cleaved by Lpro, greatly reducing host cap-dependent mRNA translation without affecting viral cap-independent protein synthesis, which is a characteristic of most picornavirus infections (de Los Santos et al., 2009; Devaney et al., 1988; Kirchweger et al., 1994; Zhu et al., 2010). Additionally, Lpro inhibits dsRNA-induced type Ⅰ interferon (IFN) transcription by inhibiting the expression of IFN-regulatory factor 3/7 (Wang et al., 2010).
In eukaryotic cells, the SAP domain (scaffold-attachment factors A and B, apoptotic chromatin-condensation inducer in the nucleus, and protein inhibitor of activated STAT proteins) is a putative DNA-binding domain found in diverse nuclear proteins (Aravind et al., 2000). The SAP domain consists of 35 amino acids, including conserved hydrophobic and charged residues (Aravind et al., 2000), and is found in a number of chromatin-associating proteins, such as scaffold-attachment factors, DNA-repair proteins, RNA-processing complexes, and proto-oncogene proteins (Ahn et al., 2003; Aravind et al., 2000; Bohm et al., 2005; Kipp et al., 2000).
A conserved SAP domain was also identified in the Lpro-coding region of FMDV (de Los Santos et al., 2009). Genetically engineered FMDV strains lacking the Lpro-coding region (leaderless viruses) or possessing a mutated Lpro SAP domain exhibited attenuated viral replication in infected cattle and swine (Chinsangaram et al., 1998; Diaz-San Segundo et al., 2012; Zhu et al., 2010). SAP-domain mutants carrying I55A and L58A substitutions abolish Lpro retention in the nuclei of FMDV-infected cells and subsequently prevent FMDV-related degradation of nuclear factor-kappa B (NF-κB) (de Los Santos et al., 2007), resulting in upregulation of several cytokines, chemokines, and IFN-stimulated genes (ISGs) (de Los Santos et al., 2009). Additionally, inoculation of swine with FMDV containing a mutated SAP domain induced early protection against disease (Diaz-San Segundo et al., 2012), and transcriptome analysis of embryonic bovine kidney cells (EBKs) infected with SAP-mutated FMDV showed enhanced expression of various IFN-related genes as compared with EBK cells infected with FMDV containing a wild-type SAP domain (de Los Santos et al., 2009).
FMDV pathogenesis presents particular features depending on the host (Pega et al., 2013). In different species, the viral entry routes, primary infective and replicative sites, and, consequently, the associated symptoms and immune responses elicited showed clear differences (Alexandersen et al., 2003). The different host responses triggered by FMDV also correlated with viral replication, thereby affecting viral propagation in different cells. In EBK cells, an intact Lpro SAP domain was correlated with type Ⅰ IFN responses (de Los et al., 2006; Zhu et al., 2010). Here, we compared the pathogenic characteristics of FMDV containing wild-type or mutant SAP domains in swine PK15 and SK6 cells and found that the SAP-mutant variant exhibited decreased pathogenicity in both cell lines, with a more pronounced decrease observed in SK6 cells, relative to the wild-type SAP variant.
To analyze the different transcription profiles induced by SAP-domain status, FMDV containing either wild-type or mutant SAP variants was used to infect SK6 cells, followed by comparative transcriptome analysis using next-generation sequencing (NGS) technology to systematically observe the differences in gene expression and host response. Of 20, 421 genes detected, differentially enhanced or repressed expression was observed in 1, 670 and 183 genes, respectively, with many associated with antiviral responses involving transcription, immune response, inflammation, apoptosis, and cytokines or chemokine production. Our results indicated that the FMDV Lpro SAP domain was significantly correlated with FMDV viral pathogenicity in SK6 cells, and that the differential expression of various host genes is dependent upon infection with FMDV containing an intact Lpro SAP domain.
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An engineered chimeric virus, rA-FMDV, which was previously constructed by Zheng et al., was used as a candidate vaccine (Zheng et al., 2013). The rA-FMDV was constructed by replacing the P1 gene in the O/CHA/99 strain (GenBank accession number: AF506822) with the P1 gene from the A/HuBWH/CHA/2009 strain (GenBank accession number: JF792355). A SAP-mutant virus, rA-SAP-FMDV, was previously constructed by Zheng et al. by introducing the I55A and L58A mutations into the SAP domain of Lbpro in rA-FMDV (unpublished data). The schematic representation of the mutation information is shown in Figure 1A. SK6 cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, USA) and cultured in the medium supplemented with 10% fetal bovine serum (FBS) at 37℃ under 5% CO2.
Figure 1. (A) Schematic of the I55A and L58A mutations (red, italics) in the Lpro SAP domain. (B) Different pathogenicity observed between rA-FMDV and rA-SAP-FMDV in SK6 and PK15 cells. SK6 and PK15 cells were infected with rA-FMDV or rA-SAP-FMDV at similar MOIs, and the viral TCID50 was detected and recorded. Results are presented as the mean ± standard error from three independent experiments.
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SK6 and PK15 cells were washed with phosphate-buffered saline and infected with FMDV at a multiplicity of infection (MOI) of 1 at 37℃. After a 1-h adsorption period, the supernatant was removed, and the cells were incubated at 37℃ with DMEM containing 0.5% FBS. The cells used for transcription profile analyses were harvested at 6-h post-infection, because a minimal cytopathic effect (CPE) was observed at ~6 h, enabling a more complete identification of differentially expressed genes. The TCID50 assay was performed in 96-well plates according to standard procedures, and cells were cultured until CPEs were clearly observed (3-5 days). The TCID50 value was calculated using the Reed-Muench method (Reed et al., 1938).
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Total RNA was extracted from SK6 cells using TRIzol Reagent (Invitrogen) according to manufacturer protocol. Two micrograms of total RNA was used to synthesize the first strand of cDNA using M-MLV reverse transcriptase (Invitrogen), and the synthesized cDNA were subjected to qPCR analysis performed using SYBR Premix Ex Taq (Takara, Kyoto, Japan) according to manufacturer protocol. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. Primer sequences used in this study are listed in Supplementary Table S1. The results were obtained from three independent experiments.
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A sample-pooling strategy was performed in this study. The rA-FMDV-and rA-SAP-FMDV-infected samples were used as mixture samples, with each sample prepared by mixing four different dishes of virus-infected cells. After total-RNA extraction and DNase Ⅰ treatment, magnetic beads conjugated with oligo (dT) were used to isolate mRNA. The mRNA was divided into short fragments, and cDNA was synthesized using the mRNA fragments as templates. The synthesized cDNA was purified and resolved with elution buffer [10 mmol/L Tris-Cl (pH 8.5)] for end repair and single-nucleotide (adenine) addition. Subsequently, the treated fragments were connected using adapters and subjected to agarose gel electrophoresis, and suitable fragments were selected as templates for PCR amplification. The quality of the obtained library was verified using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) for quantification and qualification. The library was sequenced using an Illumina HiSeqTM 2000 (Illumina, San Diego, CA, USA), and the raw data was deposited as a National Center for Biotechnology Information (NCBI) BioProject (accession reference: PRJNA269140).
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To identify the host genes associated with FMDV pathogenicity in SK6 cells, the original data were filtered and screened for differentially expressed genes. The NCBInr database was selected as the analytical database, and genetic data containing adaptors and low-quality reads were excluded. The short oligonucleotide analysis package (SOAPaligner/SOAP2) (Li et al., 2008) was used to quickly and accurately align the reads generated by the Illumina/Solexa Genome Analyzer (Illumina); and the reads per kilobase transcriptome per million mapped reads [RPKM; RPKM=106C/(N×L/103), where C represents the number of reads uniquely aligned to the gene of interest, N is the total number of reads that are uniquely aligned to all genes, and L is the number of bases in the gene of interest (Mortazavi et al., 2008)] was calculated. The RPKM method is able to eliminate the influence of different gene lengths and sequencing discrepancies on the calculation of gene expression. Therefore, the calculated gene expression can be used for comparing differences in gene expression among samples. A method to calculate the significance of digital gene-expression profiles was used for analysis of differentially expressed genes (Audic et al., 1997). We used a false discovery rate (FDR) ≤ 0.001 (mascot FDR calculation: http://www.matrixscience.com/help/decoy_help.html) and an absolute value of the Log2Ratio ≥ 1 as the thresholds to judge the significance of differences in gene expression. The screened differentially expressed genes were further analyzed by Gene Ontology (GO) and pathway-enrichment analysis. The GO database (http://www.geneontology.org/) and GO TermFinder software (http://smd.stanford.edu/help/GO-TermFinder/GO_TermFinder_help.shtml/) were used to perform GO analysis, and the Kyoto Encyclopedia of Genes and Genomes database (Kanehisa et al., 2008) was used for pathway-enrichment analysis.
Viruses and cells
Virus infection and 50% tissue culture infectious-dose (TCID50) assay
RNA extraction and real-time quantitative PCR (qPCR)
RNA library construction and Solexa/Illumina sequencing
Gene analysis
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The mutated region in the SAP domain of rA-SAP-FMDV was confirmed by sequencing analysis. A low MOI leads to infection of a percentage of cells, resulting in these infected cells signaling adjacent, uninfected cells via cytokines in order to activate antiviral genes, including secreted innate immune proteins. To study the signal transduction pathways and proteins involved, infections in this study were performed at 1 MOI. To compare the replication status of rA-FMDV and rA-SAP-FMDV in porcine PK15 and SK6 cells, the cells were infected with equal concentrations of rA-FMDV or rA-SAP-FMDV. The samples were collected 12-h post-infection, and the titers determined by TCID50 assay. The results showed that rA-FMDV replicated more quickly relative to rA-SAP-FMDV in both PK15 and SK6 cells (Figure 1B), indicating that the SAP mutation decreased FMDV replication in porcine PK15 and SK6 cells, with a larger decrease observed in SK6 cells.
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To explore the differentially expressed genes involved in the altered pathogenicity observed in SK6 cells infected with FMDV containing the SAP mutation, rA-FMDV-infected and rA-SAP-FMDV-infected SK6 cells were collected at 6-h post-infection, and transcriptome analysis was performed. After a stringent quality check and filtering of the data (FDR ≤ 0.001 and fold-change ≥ 2), 20, 421 genes were detected, with 1, 853 differentially expressed genes identified. A total of 1, 670 and 183 genes were differentially upregulated and downregulated, respectively, between rA-SAP-FMDV-and rA-FMDV-infected SK6 cells (Supplementary Figure S1, S2). The expression of 117 transcription factor-related genes involved in 12 biological processes, 114 immune regulation-related genes participating in 40 immune-regulatory processes, 69 cytokine-related genes, including 20 involved in cytokine-production and -secretion processes, 12 inflammatory response-related genes, and 19 apoptosis-related genes were significantly altered (Table 1, Supplementary Table S2-S5). The distinctively different expression profiles of these genes may explain the decreased pathogenicity observed following infection with rA-SAP-FMDV. An analysis of the available literature indicated that the majority of the differentially expressed genes correlating with antiviral responses included (Table 2): 1) genes involved in transcriptional regulation (EIF4A2, EIF5B, EIF3J, NFKBIA, and NFKBIZ); 2) genes involved in the regulation of immune response (IFIT1, ITCH, IL7R, JAK2, LTB, TNFSF10, IL7, BLM, IFIT1, IL18, IL6, and FOS); 3) cytokine-related genes (IL1, IL6, IL20, TNF, CCL2, CCL20, CXCL10, CXCL2, CCL3L1, CCL4, CCL5, and CXCL11); 4) genes involved in the regulation of inflammation and chemokine production (TNF, CCL5, IL1A, IL6, IL6ST, CCL2, and ITCH); and 5) genes involved in apoptosis (BLM, CASP3, BRCA2, PMAIP1, CD38, MAP3K5, CUL5, TNFSF10, and XIAP).
Function Total gene number Up-or down-regulated gene number Up-regulated Down-regulated transcription factor-related genes 117 109 8 immune regulation-related genes 114 104 10 cytokine-related genes 69 62 7 inflammatory response-related genes 12 11 1 apoptosis-related genes 19 18 1 Table 1. Summary of differential expressed genes
Gene Fold Gene description Function EIF4A2 2.23 Eukaryotic initiation factor 4A-Ⅱ RNA helicase activity; adenyl ribonucleotide binding EIF5B 3.11 Eukaryotic translation initiation factor 5B Translation factor activity, nucleic acid binding EIF3J 2.47 Eukaryotic translation initiation factor 3 subunit
J-like isoform 1Translation factor activity, nucleic acid binding NFKBIA 3.83 NF-kappa-B inhibitor alpha Transcription factor binding NFKBIZ 3.47 NF-kappa-B inhibitor zeta Transcription cofactor activity, protein binding IFIT1 3.74 Interferon induced protein with tetratricopeptide repeats 1 RNA binding, protein binding ITCH 2.87 Itchy E3 ubiquitin protein ligase Chemokine receptor binding, ubiquitin protein ligase activity IL7R 4.32 Interleukin 7 receptor Cytokine receptor activity JAK2 2.27 Janus kinase 2 Kinase binding, cytokine receptor, protein kinase activity LTB 13.15 Lymphotoxin-beta Tumor necrosis factor receptor superfamily bindin TNFSF10 3.67 Tumor necrosis factor superfamily member 10 Cation binding, tumor necrosis factor receptor binding IL7 2.31 PREDICTED: interleukin-7 isoform 3 Cytokine receptor binding BLM 3.15 Bloom syndrome protein ATP-dependent DNA helicase activity, double-stranded DNA binding IL18 2.27 Interleukin 18 Receptor binding, cytokine activity IL6 11.05 Interleukin 6 Cytokine receptor binding, cytokine activity FOS 3.78 FBJ osteosarcoma oncogene Nucleic acid binding transcription factor activity, protein dimerization activity IL1 3.11 Interleukin 1 Cytokine activity IL20 27.75 Interleukin 20 Cytokine receptor binding, cytokine activity TNF 20.89 Tumor necrosis factor Tumor necrosis factor receptor superfamily binding, sequence-specific DNA binding CCL2 5.73 C-C motif chemokine ligand 2 Kinase activity, chemokine receptor binding CCL20 7.62 C-C motif chemokine ligand 20 Cytokine activity, chemokine receptor binding CXCL10 5.89 Chemokine (C-X-C motif) ligand 10 Protein kinase regulator activity, cytokine activity CXCL2 2.38 Chemokine (C-X-C motif) ligand 2 Cytokine activity, chemokine activity CCL3L1 6.17 C-C motif chemokine ligand 3 like 1 CCR chemokine receptor binding, chemokine activity CCL4 15.34 C-C motif chemokine ligand 4 Cytokine activity, chemokine activity CCL5 2.58 C-C motif chemokine ligand 5 CCR chemokine receptor binding, chemokine activity, protein tyrosine kinase activator activity CXCL11 6.17 C-X-C motif chemokine ligand 11 Heparin binding, chemokine activity IL1A 3.11 Interleukin-1 alpha precursor Transition metal ion binding, cytokine receptor binding IL6ST 3.58 Interleukin 6 signal transducer Ciliary neurotrophic factor receptor activity, cytokine receptor binding CASP3 2.08 Caspase 3 Endopeptidase activity, cyclin-dependent protein kinase regulator activity BRCA2 5.42 Breast cancer 2 Structure-specific DNA binding, histone acetyltransferase activity PMAIP1 2.78 Phorbol-12-myristate-13-acetate-induced protein 1 Protein binding CD38 2.51 Cluster of differentiation 38 Transferase activity, NAD(P)+ nucleosidase activity MAP3K5 2.23 Mitogen-activated protein kinase kinase kinase 5 Metal ion binding, phosphatase binding, apoptotic protease activator activity CUL5 3.23 Cullin 5 Signal transducer activity, enzyme binding XIAP 2.59 X-linked inhibitor of apoptosis Transition metal ion binding, cysteine-type endopeptidase inhibitor activity Note: A minimum of twofold change (P < 0.0001, Q < 0.0001) was used as the standards for selecting genes of interest. Table 2. List of genes that displayed significant differential expression at WT and SAP mutant FMDV-infected SK6 cells
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To further confirm and validate the transcriptome analysis results, we performed qPCR analysis to determine the reproducibility of the differential gene expression. A selected group of genes for which we had an established method available in our laboratory (with established primers and melting/annealing temperatures previously) were chosen for analysis. Six upregulated genes (CCL4, CCL2, IL6, IL7, IL18, and EGR1) and four downregulated genes (SRPX2, CREB5, RASAL1, and RIN2) were analyzed, and qPCR results confirmed the differential expression identified between rA-SAP-FMDV-and rA-FMDV-infected cells. As shown in Figure 2, the qPCR results corresponded with transcriptome analysis results and, while some fold-change differences were observed between results from each method, similarities in the overall expression profiles were revealed.
Figure 2. Validation of differentially expressed genes identified by transcriptome analysis through qPCR detection. Six upregulated and four downregulated genes were detected in an independent infection experiment undertaken in order to validate transcriptome analysis results. The expression profiles of the 10 selected genes were consistent between the transcriptome-analysis and qPCR-detection results. Results are presented as the mean ± standard error from three independent experiments.
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Infection with FMDV containing the SAP-domain mutation altered gene expression in SK6 cells, thereby affecting various biological processes and signal transduction pathways, and resulting in blocked viral replication and decreased pathogenicity. To systematically analyze the functional characterization of the differentially expressed genes and pathways associated with the FMDV Lpro SAP domain, GO analysis and pathway annotation were conducted. The results indicated that the differentially expressed genes were involved in metabolism, cell cycle processes, and cellular component organization or biogenesis processes (Supplementary Figure S3), and functional analysis revealed that many of these genes were involved in nucleotide binding and functions associated with nucleic acids (Supplementary Figure S4). Cellular component annotation results are shown in Supplementary Figure S5.
Pathway analysis indicated that 35 pathways were altered in rA-SAP-FMDV-infected cells as compared with rA-FMDV-infected cells, including regulation of actin cytoskeleton formation, endocytosis, phagosome formation, chemokine-signaling pathways, the cell cycle, the retinoic acid-inducible gene 1-like receptor signaling pathway, the NF-κB signaling pathway, and the nucleotide-binding oligomerization domain-like receptor signaling pathway (Supplementary Figure S6). The potential host targets for Lpro and the protein-protein interaction pathways involved are shown in Figure 3. These findings suggested that expression of these genes might potentially result in decreased rA-SAP-FMDV replication.
Viral replication in porcine cells differs between FMDV containing wild-type or mutant SAP domain
Differentially expressed genes between SK6 cells infected with FMDV containing wild-type or mutant SAP domain
Validation of differentially expressed genes by qPCR
Functional characterization of differentially expressed genes and pathways affected by infection with FMDV containing the SAP mutation
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FMDV has the ability to manipulate various host-cell signal transduction pathways by subverting gene expression. FMDV Lpro is a host-cell antagonist that interferes with host gene expression, promotes viral propagation, and subverts host immune systems by targeting eIF-4G, IRF3, IRF7, and NF-κB (de Los Santos et al., 2009; Kirchweger et al., 1994; Wang et al., 2010). The FMDV Lpro SAP domain is a putative DNA-binding domain found in diverse eukaryotic nuclear proteins (Aravind et al., 2000; de Los Santos et al., 2009) and inhibits host innate immune response (de Los Santos et al., 2009). The mutation of two amino acids (I55A and L58A) in the Lbpro SAP domain alters Lpro subcellular localization and function (de Los Santos et al., 2009; Diaz-San Segundo et al., 2012).
In a previous study, we constructed a chimeric virus, rA-FMDV, as a candidate vaccine (Zheng et al., 2013). To decrease rA-FMDV pathogenicity and develop a potential live attenuated vaccine, we constructed rA-SAP-FMDV containing a mutated SAP domain (unpublished data). Here, we compared the different pathogenic characteristics of rA-FMDV and rA-SAP-FMDV in PK15 and SK6 cells, and showed that rA-SAP-FMDV was less pathogenic relative to rA-FMDV in both cell lines, suggesting that rA-SAP-FMDV may have potential as a vaccine strain based on its failure to disrupt cellular responses that inhibit viral replication.
Decreased rA-SAP-FMDV replication was more evident in SK6 cells as compared with PK15 cells. To analyze the differentially expressed genes correlated with the altered viral replication observed in SK6 cells, transcriptome analysis was performed using NGS technology. The results indicated differential expression of 1, 853 genes between infected and non-infected SK6 cells, with these findings subsequently confirmed by qPCR analysis. These findings suggested that mutation of the FMDV Lpro SAP domain might adversely affect the ability of FMDV to inhibit host gene expression during infection, resulting in reduced viral pathogenicity.
Among the differentially expressed genes upregulated in rA-SAP-FMDV-infected cells, EIF4A2, EIF5B, and EIF3J are involved in the initiation of host translation by aiding in the recruitment of protein and mRNA components to ribosomes (Cheyssac et al., 2006; ElAntak et al., 2007; Kyono et al., 2002; Meijer et al., 2013; Unbehaun et al., 2007). Swine infected with FMDV containing a mutated SAP domain developed a strong neutralizing-antibody response as early as 2-days post-inoculation as compared with those infected with wild-type FMDV (Diaz-San Segundo et al., 2012). The upregulation of these translation factors possibly resulted in the enhancement of neutralizing-antibody production. Furthermore, our analysis revealed upregulation of other genes, including those involved in metabolic and cellular-response processes (Supplementary Figure S3).
FMDV infection can induce degradation of NF-κB (de Los Santos et al., 2007); however, NF-κB activity was significantly enhanced in cells infected with FMDV containing a mutated SAP domain (Zhu et al., 2010). NFKBIA and NFKBIZ are involved cytokine production through NF-κB regulation (Ninomiya-Tsuji et al., 1999; Yamazaki et al., 2001). In this study, we found that the expression of NFKBIA and NFKBIZ was upregulated in rA-SAP-FMDV-infected cells, which may have altered the subsequent expression of NF-κB-induced cytokines to ensure a robust immune response (Figure 3). CCL4 and IL7, both involved in cellular immune and inflammatory responses, were also upregulated in these cells. Additionally, the upregulation of many other genes involved in immune response, inflammation, chemokine production, and apoptosis was also observed.
Upregulation of EGR1 and IL6 expression was also observed in rA-SAP-FMDV-infected cells. EGR1 mediates cell proliferation, differentiation, inflammation, and apoptosis (Han et al., 2015), and may interact with p53 or FOS to regulate apoptosis or transcription (Figure 3). IL6 is involved in inflammation, B cell maturation, and suppression of viral replication (Dienz et al., 2012). Here, upregulation of IL6 expression possibly enhanced acute-phase response and suppressed rA-SAP-FMDV replication. Our results suggested that Lpro may attenuate EGR1 and IL6 expression and adversely affect regulation of cell proliferation, transcription, differentiation, inflammation, immune response, and apoptosis to promote viral replication. Furthermore, mutation of the Lpro SAP domain might impair this antagonistic effect, thereby inhibiting viral replication (Figure 3). The resulting upregulation of these genes likely reinforced the antiviral activity of the cells, directly resulting in the decreased pathogenicity observed following rA-SAP-FMDV infection. Conversely, downregulation of SRPX2, which is involved in anti-apoptotic activity, was observed in rA-SAP-FMDV-infected SK6 cells (Figures 2 and 3). This indicated an alternative pathway for suppressing viral replication through the promotion of cell death. Intact Lpro likely inhibits the initiation of apoptosis, with our results suggesting that mutation of the Lpro SAP domain impaired this inhibitory effect.
A previous study found that in EBK cells infected with a FMDV containing the SAP-domain mutation, NF-κB was the primary factor responsible for the differential transcription of many upregulated genes associated with innate immune response (Zhu et al., 2010). Here, we found that the differential gene expression observed in rA-SAP-FMDV-infected SK6 cells resulted from altered expression of genes involved in transcription and immune-related regulation. However, IFN-stimulated genes, such as ISG15, ISG20, MX1, GBP1, and OAS1, which were differentially expressed in EBK cells infected with a FMDV containing the SAP-domain mutation, were not observed in this study. This is possibly due to SK6 cells being deficient in type Ⅰ IFN production (Ruggli et al., 2003), although the enhancement of various type Ⅰ IFN-independent genes and pathways determined in this study were implied to perform crucial antiviral effects.
In summary, we reported that a FMDV Lpro SAP-domain mutant exhibited decreased replication ability in SK6 cells as compared with wild-type FMDV. Transcriptome analysis suggested that the altered expression of genes involved in transcription, immune response, cytokine and chemokine production, inflammation, and apoptosis were the primary reasons for the observed decrease in pathogenicity. Our results provided insight into the pathogenic mechanisms associated with the FMDV Lpro SAP-domain and suggested that mutation of region provides a strategy for the development new FMDV-related vaccines having impaired host-antagonistic ability.
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We thank Dr. Jinwen Liu for providing valuable technical assistance and suggestions. This work was supported by grants from the National Science and Technology Ministry (2015BAD12B04), National Natural Sciences Foundation of China (No. 31302118, 31502042 and 31402179), the Gansu Science Foundation for Distinguished Young Scholars (no. 145RJDA328), the International Atomic Energy Agency (16025/R0) and the Key technologies R & D program of Gansu Province (1302NKDA027).
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The authors declare that they have no conflict of interest. This article does not contain any studies with human oranimal subjects performed by any of the authors.
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ZXN, FY, ZXZ and HXZ designed the research, ZXN, FY, ZXZ, XD, WWL and WJC performed the experiments. XLZ, YJ, JHG and XTL provided experiment support. ZXN, ZXZ and HXZ wrote the manuscript. All authors have read and approved the final manuscript for submission.
Supplementary figures/tables are available on the website of Virologica Sinica: www.virosin.org; link.springer. com/journal/12250.
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Gene Primers(5′→3′) CCL4 Forward: CACCTCCTGCTGCTTCACATA Reverse: CAGACCTGCCTGCCCTTTT CCL2 Forward: GTCACCAGCAGCAAGTGTCCT Reverse: ATGTGCCCAAGTCTCCGTTTA IL6 Forward: GACAAAGCCACCACCCCTAA Reverse: CTCGTTCTGTGACTGCAGCTTATC IL7 Forward: GGGATGGATGAAACAGAAGG Reverse: GCTACTGGCAACAGAACAAGG' IL18 Forward: GCACCTCAGACCGTATTTATT Reverse: CATCATGTCCAGGAACACTTC EGR1 Forward: TCAACACCACCTACCAGTCCCA Reverse: GATCTTGGTATGCCTCTTGCGTT SRPX2 Forward: AACGTGGTATGCAGGTTCAGG Reverse: GTAGTCACAGCGGGAGTCAAGA CREB5 Forward: TATCTTCCCTGCTACATCTTCACA Reverse: AACGCAGCCTTCAACCTCATT RASAL1 Forward: GTGAAAGTGGACGACGAGGTGG Reverse: GGGAAGCGTGTCTTCTTGATGG RIN2 Forward: CCTTGAAGTTGCCTTATGCTGTTT Reverse: GCTACGTTCCCATGTGGGTGAT GAPDH Forward: ACATGGCCTCCAAGGAGTAAGA Reverse: GATCGAGTTGGGGCTGTGACT Table S1. The qPCR primers used in this study
Gene function description Up-regulated genes Down-regulated genes Gene number Gene name list Gene number Gene name list Transcription factor complex 9 ING2, CNOT7, TAF13, TAF9B, MNAT1, RB1, NFYB, GTF2A1, TAF2 0 - Transcription factor binding 22 HES1, KLF4, LOC100154750, GTF2A1, LOC100739605, MDFIC, MTDH, MED13, JMJD1C, PRDM5, LOC100626982, NFYB, EGR2, NRIP1, NFKBIA, SIRT1, HMGB1, CAND1, EIF4A2, EIF4A2, EIF5B, EIF3J 0 - Protein binding transcription factor activity 34 FGF2, JMY, SP100, IFNB1, NPAT, MTDH, CIR1, SCAI, USP16, NFE2, GABPA, SIRT1, C1D, LOC102161761, LOC102160069, TRIP11, LOC100154750, MYSM1, RB1, SS18, LOC100739605, LOC100622863, TAF9B, TMF1, MED13, NMI, COPS2, LOC102166221, TBL1XR1, CASP8AP2, NRIP1, AEBP2, KMT2E, SKIL 3 DYRK1B, PIR, TFCP2L1 Transcription factor binding transcription factor activity 34 FGF2, JMY, SP100, IFNB1, NPAT, MTDH, CIR1, SCAI, USP16, NFE2, GABPA, SIRT1, C1D, LOC102161761, LOC102160069, TRIP11, LOC100154750, MYSM1, RB1, SS18, LOC100739605, LOC100622863, TAF9B, TMF1, MED13, NMI, COPS2, LOC102166221, TBL1XR1, CASP8AP2, NRIP1, AEBP2, KMT2E, SKIL 3 DYRK1B, PIR, TFCP2L1 Nucleic acid binding transcription factor activity 60 KLF4, ZHX1, EGR4, ZNF24, LOC102159255, TFAM, NFIA, EGR2, GABPA, GATA3, NFE2L2, HDAC1, HDAC2, HES1, PAXBP1, ZC3H8, TAF13, CREBRF, GATA2, TOPOⅡ, TOP2B, RBPJ, MYNN, EHF, DMTF1, ZNF84, ZBTB38, AHR, HMGB1, ZNF197, CNOT7, GCFC2, BTAF1, LOC102163244, SLC30A9, NPAT, LOC100737142, CIR1, ZNF189, ETV1, FOXN2, TBX21, SIX4, ZEB2, NFYB, EGR1, ZNF287, ARID4A, RB1, BTG2, FOS, LOC100622863, BLZF1, LOC100520527, LOC100515279, NR1D2, SCML2, HIF1A, LOC100626982, POU3F2 5 ARNT2, NR4A2, RCAN1, ZNF71, TFCP2L1 Ligand-activated sequence-specific DNA binding RNA polymerase Ⅱ transcription factor activity 1 NR1D2 1 NR4A2 Sequence-specific DNA binding transcription factor activity 5 HES1, BTG2, HIF1A, NR1D2, FOXN2 1 NR4A2 Sequence-specific DNA binding RNA polymerase Ⅱ transcription factor activity 4 BTG2, HIF1A, NR1D2, FOXN2 1 NR4A2 Positive regulation of sequence-specific DNA binding transcription factor activity 8 MALT1, TNF, KRAS, TAB3, CHUK, LOC100620995, NFKBIA, MTDH 0 - Regulation of sequence-specific DNA binding transcription factor activity 10 MALT1, ITCH, TNF, KRAS, TAB3, CHUK, LOC100620995, NFKBIA, LOC100153617, MTDH 1 LOC100515993 Negative regulation of sequence-specific DNA binding transcription factor activity 3 NFKBIA, LOC100153617, ITCH 1 LOC100515993 Regulation of transcription factor import into nucleus 2 NFKBIA, TNF 0 - Table S2. Differentially expressed genes involved in transcription-related functions
Gene function description Up-regulated genes Down-regulated genes Gene number Gene name list Gene number Gene name list Negative regulation of immune effector process 4 SOCS5, IFIT1, ITCH, IL7R 1 TGFB3 Production of molecular mediator of immune response 8 MALT1, LOC100621191, TNF, XRCC4, LOC100620995, LOC100739713, IL7R, MSH2 0 - Immune system process 94 BMPR1A, IL18, CASP3, CXCL10, SEMA3C, TGFBR1, MASP2, IFNB1, IFIT1, CCL5, ROCK1, S100A12, LOC100739781, LOC100625180, LOC100518921, CD79A, XRCC4, CD84, GATA3, IL7R, DPP8, HES1, LOC102161761, ZC3H8, CSF2, TNF, TBK1, TAB3, PIK3CA, IRG6, LOC100736872, RBPJ, BRCA2, ATM, CCL3L1, LOC100620995, AHR, HMGB1, APC, MSH2, SLAMF7, CCL20, S100A9, ADAM10, KRAS, JAK2, LTB, TNFSF10, IL7, BLM, LOC100622217, IL1RAP, SIX4, LOC100622970, LOC100738058, RTKN2, NCK1, MAP3K8, EGR1, CHUK, IL1A, LOC100739713, ANGPT1, KITLG, LOC102163753, LOC100621191, LOC100627112, RB1, MSH3, CXCL2, UBD, BMI1, FOS, LOC100628215, STXBP3, MALT1, CCL2, MTAP, TNFSF15, CFH, CSF1, CD38, APOBEC1, NFKBIA, RICTOR, CCL4, KMT2E, CXCL11, ITGA6, LOC100739007, IL6ST, SKIL, IL6, LOC100620730 6 SUSD2, GRB7, MAP2K6, GPX2, LOC102166152, LOC100522330 Cytokine production involved in immune response 4 MALT1, LOC100620995, LOC100621191, TNF 0 - Somatic recombination of immunoglobulin genes involved in immune response 3 LOC100739713, MSH2, XRCC4 0 - Somatic diversification of immunoglobulins involved in immune response 3 LOC100739713, MSH2, XRCC4 0 - Immunoglobulin production involved in immunoglobulin mediated immune response 3 LOC100739713, MSH2, XRCC4 0 - Regulation of immune system process 48 LOC100737466, IL18, CASP3, ITCH, ADAM10, MASP2, IFNB1, IFIT1, CCL5, EDN1, IL7, BLM, LOC100622217, CD79A, TBX21, LOC100622970, LOC100738058, NCK1, MAP3K8, CHUK, IL7R, SOCS5, LOC100627112, LOC100737558, TNF, TBK1, RB1, TAB3, PIK3CA, FOS, ELMOD2, LOC100628215, MALT1, CCL2, HIF1A, ATM, ACVR2A, CFH, CSF1, CD38, RICTOR, LOC100620995, NFKBIA, KMT2E, DDX58, LOC100739007, IL6, APC 5 TNFSF4, MAP2K6, SEMA7A, TGFB3, LOC102161418 Somatic diversification of immune receptors via germline recombination within a single locus 5 LOC100739713, HMGB1, MSH3, MSH2, XRCC4 0 - Immune response 19 IL18, LOC100621191, TNF, MASP2, IFNB1, IFIT1, BMI1, IL7, STXBP3, S100A12, MALT1, CCL2, CFH, XRCC4, LOC100620995, LOC100739713, GATA3, IL6ST, MSH2 1 GPX2 Adaptive immune response 10 MALT1, IL18, TNF, MASP2, XRCC4, LOC100620995, LOC100739713, GATA3, IL6ST, MSH2 0 - Somatic diversification of immune receptors 5 LOC100739713, HMGB1, MSH3, MSH2, XRCC4 0 - Immune system development 29 KITLG, ZC3H8, RB1, MSH3, TGFBR1, BMI1, JAK2, LTB, RBPJ, BLM, LOC100628215, LOC100739781, MALT1, BRCA2, ATM, LOC100518921, CD79A, SIX4, MTAP, RTKN2, XRCC4, EGR1, CHUK, LOC100739713, HMGB1, IL7R, APC, ANGPT1, MSH2 ; 1 LOC102166152 Myeloid cell activation involved in immune response 2 S100A12, STXBP3 0 - Somatic diversification of immune receptors via somatic mutation 2 LOC100739713, MSH2 0 - Adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains 9 MALT1, IL18, TNF, MASP2, XRCC4, LOC100620995, LOC100739713, GATA3, MSH2 0 - Regulation of production of molecular mediator of immune response 2 TBX21, TNF 2 SEMA7A, TGFB3 Negative regulation of immune system process 5 CASP3, SOCS5, IFIT1, ITCH, IL7R 1 TGFB3 Regulation of immune effector process 11 LOC100737466, SOCS5, TBX21, ITCH, TNF, IFNB1, IFIT1, DDX58, ELMOD2, IL6, IL7R 2 TGFB3, SEMA7A Immunoglobulin mediated immune response 5 LOC100739713, TNF, MASP2, MSH2, XRCC4 0 - Positive regulation of immune system process 28 IL18, SOCS5, LOC100627112, ITCH, TBK1, ADAM10, TAB3, MASP2, IFNB1, PIK3CA, FOS, CCL5, EDN1, BLM, LOC100622217, TBX21, CD79A, CFH, LOC100622970, LOC100738058, NCK1, MAP3K8, CD38, RICTOR, CHUK, LOC100620995, NFKBIA, IL6 2 TNFSF4, MAP2K6 Regulation of cytokine production involved in immune response 0 - 2 SEMA7A, TGFB3 Innate immune response 2 CCL2, IFIT1 0 - Activation of innate immune response 8 LOC100622970, TBK1, TAB3, LOC100738058, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6 Innate immune response-activating signal transduction 8 LOC100622970, TBK1, TAB3, LOC100738058, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6 Immune effector process 14 S100A12, STXBP3, MALT1, CFH, TNF, MASP2, XRCC4, APOBEC1, LOC100620995, IRG6, KMT2E, LOC100739713, GATA3, MSH2 0 - Positive regulation of innate immune response 8 LOC100622970, TBK1, TAB3, LOC100738058, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6 Regulation of innate immune response 8 LOC100622970, TBK1, TAB3, LOC100738058, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6 Regulation of immune response 17 SOCS5, TBK1, TAB3, MASP2, PIK3CA, FOS, TBX21, CD79A, LOC100622970, CFH, LOC100738058, NCK1, CD38, CHUK, LOC100620995, NFKBIA, IL6 3 TGFB3, MAP2K6, SEMA7A Immune response-activating cell surface receptor signaling pathway 7 NCK1, CD38, PIK3CA, NFKBIA, LOC100620995, CHUK, CD79A 0 - Immune response-regulating cell surface receptor signaling pathway 7 NCK1, CD38, PIK3CA, NFKBIA, LOC100620995, CHUK, CD79A 0 - Activation of immune response 14 CD79A, CFH, LOC100622970, TBK1, TAB3, MASP2, LOC100738058, NCK1, CD38, PIK3CA, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6 Immune response-activating signal transduction 12 CD79A, LOC100622970, TBK1, TAB3, LOC100738058, NCK1, CD38, PIK3CA, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6 Immune response-regulating signaling pathway 12 CD79A, LOC100622970, TBK1, TAB3, LOC100738058, NCK1, CD38, PIK3CA, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6 Positive regulation of immune response 14 CD79A, CFH, LOC100622970, TBK1, TAB3, MASP2, LOC100738058, NCK1, CD38, PIK3CA, CHUK, LOC100620995, NFKBIA, FOS 1 MAP2K6 Humoral immune response mediated by circulating immunoglobulin 2 TNF, MASP2 0 - Humoral immune response 3 CFH, TNF, MASP2 0 - Cell activation involved in immune response 2 S100A12, STXBP3 0 - Leukocyte activation involved in immune response 2 S100A12, STXBP3 0 - Positive regulation of immune effector process 3 SOCS5, TBX21, IL6 0 - Table S3. Differentially expressed genes involved in immune regulation
Gene function description Up-regulated genes Down-regulated genes Gene number Gene name list Gene number Gene name list Cytokine receptor binding 25 KITLG, CSF2, IL20, ITCH, TNF, TGFBR1, IFNB1, CCL5, JAK2, LTB, TNFSF10, IL7, LOC100739781, CCL2, IL1RAP, TNFSF15, CSF1, CASP8AP2, IL1A, LIFR, LOC100739007, IL6ST, IL6, LOC100620730, ANGPT1 3 TGFB3, TNFSF4, LOC100515993 Cytokine activity 9 CCL2, CCL20, CXCL10, CXCL2, CCL3L1, CCL4, CCL5, CXCL11, LOC100620730 0 - Cytokine receptor activity 5 IL1RAP, LIFR, IL12RB2, IL6ST, IL7R 0 - Positive regulation of cytokine production 18 CSF2, LOC100737466, IL18, TNF, TBK1, IFNB1, POLR3G, NOX1, IL12RB2, MALT1, HIF1A, LOC100622970, LOC100620995, IL1A, DDX58, GATA3, IL6, IL6ST 1 TNFSF4 Regulation of cytokine production 26 CSF2, LOC100737466, IL18, ITCH, TNF, TBK1, IFNB1, IFIH1, POLR3G, NOX1, IL12RB2, LTB, RNF125, ATG5, MALT1, HIF1A, LOC100625180, TNFSF15, LOC100622970, ZNF287, LOC100620995, IL1A, GATA3, DDX58, IL6ST, IL6 5 LOC780431, TGFB3, TNFSF4, SEMA7A, ACP5 Response to cytokine stimulus 17 CASP3, SP100, TNF, ADAM10, UBD, IFIT1, IFIT3, RPS6KB1, EDN1, ACSL4, CCL2, IFIT2, EGR1, CD38, LIFR, HMGB1, IL6 2 LOC100515993, LOC102161418 Cytokine production involved in immune response 4 MALT1, LOC100620995, LOC100621191, TNF 0 - Regulation of cytokine secretion 3 IL1A, TNFSF15, TNF 1 TNFSF4 Negative regulation of cytokine production 9 ATG5, LOC100737466, ITCH, LOC100622970, TNF, TBK1, IFIH1, DDX58, RNF125 3 TGFB3, TNFSF4, ACP5 T cell cytokine production 2 MALT1, LOC100620995 0 - Cytokine production 9 MALT1, IL18, HIF1A, IL1RAP, LOC100621191, TNF, LOC100620995, IL12RB2, JAK2 1 ACP5 Negative regulation of cytokine-mediated signaling pathway 2 CCL5, IL6ST 0 - Negative regulation of response to cytokine stimulus 2 CCL5, IL6ST 0 - Cytokine-mediated signaling pathway 9 EGR1, CCL2, IFIT1, LIFR, IFIT3, SP100, IL6, TNF, IFIT2 1 LOC102161418 Cellular response to cytokine stimulus 9 EGR1, CCL2, IFIT1, LIFR, IFIT3, SP100, IL6, TNF, IFIT2 1 LOC102161418 Positive regulation of cytokine biosynthetic process 6 LOC100625180, IL1A, LTB, LOC100622970, TNF, TBK1 0 - Regulation of cytokine production involved in immune response 0 - 2 SEMA7A, TGFB3 Regulation of cytokine biosynthetic process 7 LOC100625180, IL1A, LTB, IL6, LOC100622970, TNF, TBK1 0 - Regulation of cytokine-mediated signaling pathway 4 SOCS1, CCL5, JAK2, IL6ST 0 - Regulation of response to cytokine stimulus 4 SOCS1, CCL5, JAK2, IL6ST 0 - Table S4. Differentially expressed genes involved in cytokine-related functions
Gene function description Up-regulated genes Down-regulated genes Gene number Gene name list Gene number Gene name list Chronic inflammatory response 8 TNF, PTGER3, PLA2G4A, CCL5, IL1A, IL6, IL6ST, PTGS2 0 - Acute inflammatory response 11 HIF1A, TNF, PTGER3, IFNB1, PLA2G4A, CCL5, IL1A, HMGB1, IL6, IL6ST, PTGS2 1 GPX2 Inflammatory response 1 TNF 1 GPX2 Inflammatory response to antigenic stimulus 8 TNF, PTGER3, PLA2G4A, CCL5, IL1A, IL6, IL6ST, PTGS2 0 - Chemokine receptor binding 4 CCL2, CCL5, ITCH, LOC100620730 0 - CCR chemokine receptor binding 2 CCL2, CCL5 0 - Regulation of execution phase of apoptosis 5 BRCA2, LOC100624979, LOC100739713, PMAIP1, MSH2 0 - Induction of apoptosis 13 CD38, LOC100738156, LOC100622580, MAP3K5, CUL5, TNFSF10, XIAP, TNF, BLM, BCLAF1, CASP3, CASP4, CASP8AP2 1 LOC102161387 Table S5. Differentially expressed genes involved in inflammation, chemokine production-, and apoptosis-related functions
Figure S1. GO analysis of the immune-related genes indicating altered expression in rA-SAP-FMDV-infected SK6 cells as compared with rA-FMDV-infected SK6 cells. The X-axis indicates the correspondent number of genes.
Figure S2. GO analysis of the transcription factor-, inflammatory response-, apoptosis-and cytokine-related genes showing altered expression in rA-SAP-FMDV-infected SK6 cells as compared with rA-FMDV-infected SK6 cells. The X-axis indicates the correspondent number of genes.
Figure S3. Biological processes derived from GO annotations for the differentially expressed genes. The X-axis indicates the correspondent number of genes.
Figure S4. Molecular functions derived from the GO annotation for differentially expressed genes. The X-axis indicates the correspondent number of genes.