Background
Porcine Epidemic Diarrhea (PED) is an acute and highly contagious enteric disease characterized by severe watery diarrhea, dehydration, and anorexia. Deceased piglets presented with thin and almost transparent small intestines containing undigested milk curdles. The etiological agent PED virus (PEDV) was first isolated and recognized from Europe in the 1970s [
1,
2], then it was spread and prevalent in Asian for decades [
3,
4]. PEDV originally caused a relatively mild and sporadic disease. However, since more virulent variant strains appeared in 2010 [
5‐
8], PEDV has been subsequently associated with severe outbreaks of diarrheal disease [
9] in Asia and in North American [
10‐
13]. Acute PEDV outbreaks normally resulted in enormous economic losses to swine industries around the world, for instance, in 2013 to 2014 PEDV killed more than 7 million pigs in the North American [
14]. Currently, PEDV poses a serious threat to the swine industry worldwide.
PEDV is an enveloped single-stranded and positive-sense RNA virus, belongs to the genus Alpha coronavirus, family Coronaviridae, order Nidovirales [
2]. The genome of PEDV is about 28 kb and comprises of a 5′ untranslated region (UTR), at least 7 open reading frames (ORF1a, ORF1b, and ORF2–6), and a 3’ UTR. The ORF 1a and 1b cover the 5′-proximal two-thirds of the genome coding for replicase polyprotein (pp) la and pp1ab, respectively [
15,
16]. These pp1a and pp1ab can be cleaved by internal proteases generating 16 nonstructural proteins, namely nsp1–16. ORF2–6 encode four structural proteins including the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins, while ORF3 encodes an accessory protein [
15]. The large spikes on the coronavirus envelope are composed of trimers of the spike proteins. The spike protein mediates viral entry into host cells by functioning as a class I viral fusion protein [
17]. During maturation, the spike protein is often cleaved into a receptor-binding subunit S1 and a membrane-fusion subunit S2 [
18].
PEDV induce infected host cell apoptosis has been established in vitro and in vivo [
19]. PEDV also induces caspase-independent apoptosis in host cells by the activation of mitochondrial apoptosis-inducing factor based on the observation of AIFM1 relocated into the nucleus during the PEDV infection [
19]. However, the protein that is mainly responsible for this induction remains unclear. In this study, we have provided evidence that S1 protein is the main inducer for cell apoptosis during PEDV infection.
Methods
Cells and viruses
Vero, Vero-E6, MARC-145 and BHK-21 cells were obtained from American Type Culture Collection (ATCC), all the cells were maintained in Dulbecco Modified Eagle Medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml of penicillin and 100 μg/ml of streptomycin) in a 5% CO2 incubator. PEDV SM98 and CV777 strains were kept in our laboratory, wild-type strains BJ2011 was isolated from a farm in Beijing in 2011.
Reagents
Fluorescein (FITC)-conjugated affiniPure Goat anti-Mouse IgG (H + L) was a product of Jackson Immuno Research; Anti-PEDV Spike mAb, Annexin V-PE/7-AAD Apoptosis Detection Kit was purchased from BD; Caspase-3, caspase-8, caspase-9 and PARP rabbit mAbs were bought from Cell Signaling Technology; Anti-AIFM1 mice mAb was a product of Sigma-Aldrich; Caspase-3, − 8 and − 9 activity assay kits were purchased from Biovision.
Plasmids construction
The Nsp1–16, M, N, E, ORF3, S1 and S2 genes were cloned from PEDV strain SM98 using specific primers (Additional file
1: Table S1). Primers were synthesized by BGI Tech Solutions Co., Ltd. (Beijing, China). All the plasmids were constructed by standard recombined DNA techniques. Briefly, all the non-structural and structural genes were amplified by RT-PCR from PEDV RNA genome and cloned into the pEGFP-N1 vector. The S1 genes of transmissible gastroenteritis virus (TGEV), avian infectious bronchitis virus (IBV) and canine coronavirus virus (CCoV) were cloned from vaccine strains (TGEV H Strain, IBV-H52 strain and CCoV K378 strain, respectively). The S1 genes of Middle East respiratory syndrome (MERS) coronavirus and Severe acute respiratory syndrome (SARS) coronavirus were synthesized by Genscript according to the sequences submitted in Genebank (accession number: KY581693.1 and AH013709.2, respectively).
Apoptosis assay
Vero, Vero-E6 and Marc-145 cells were seeded in six-well-plate and cultured overnight, cells were then mock-infected or infected with PEDV CV777 or SM98 strains at a multiplicity of infection (MOI) of 0.1 for 48 h. Cells were trypsinized and resuspended with buffer (135 mM NaCl, 10 mM HEPES, 5 mM CaCl2) and stained with Annexin V-PE and 7-AAD at room temperature for 15 min. Cells were analyzed by flow cytometry. Fluorescence-activated cells sorter (FACS) data were analyzed using CellQuest software (BD).
Vero-E6 or BHK21 cells were cultured in six-well-plate and grow to 70–80% confluent, cells were transfected with various plasmids including pEGFP-N1, pEGFP-Nsp1–16, pEGFP-M, pEGFP-N, pEGFP-E, pEGFP-ORF3, pEGFP-S1 and pEGFP-S2 by Lipofectamine LTX. After 48 h transfection, cells were harvested and stained with Annexin V-PE and 7-AAD at room temperature for 15 min. GFP-positive cells were gated for apoptosis analysis. FACS data were analyzed with CellQuest software (BD).
Western blotting
Protein samples were separated by SDS-PAGE and were electrically transferred onto a polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% skim milk in phosphate-buffered saline (PBS), the membrane was incubated with proper antibodies and subsequently probed with appropriated horseradish peroxidase (HRP) conjugated goat anti-mouse secondary antibody. The protein bands were developed with the ECL western blotting system (Fisher Scientific) and exposed to a fluorchem E apparatus (Proteinsimple, Santa Clara, CA, USA). Western blotting bands are quantified according to intensity by using ImageJ software, normalized to β-actin and expressed relative to mock infection.
Confocal microscopy assays
Vero cells were grown on coverslips in 24-well-plate (Costar, Corning Incorporation) and infected with the SM98 virus at an MOI of 0.1, uninfected cells serve as a mock control. The cells were fixed and permeabilized with cold anhydrous ethanol for 20 min at room temperature (RT) at 24, 48 and 72 h post infection, respectively. Followed by being blocked with 2% BSA in PBS for 1 h at RT. Then, the cells were incubated with mice anti-PEDV S monoclonal antibody at 4 °C in a humid chamber. After being rinsed 3 times with PBS, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mice antibody for 1 h at RT. Finally, the cells were stained with DAPI, and the images were viewed under an Olympus confocal microscope (fluoview 1000×).
Caspase-3, − 8 and − 9 activity assays
Vero cells were seeded on six-well-plate and were mock-infected or infected with SM98 strain at an MOI of 1. Infected cells were incubated for 48 h before trypsinized. Cells were washed with cold PBS, resuspended in 50 μL of chilled cell lysis buffer, and incubated on ice for 15 min. The cell lysates were centrifuged at 10,000×g for 15 min. The supernatants were collected and frozen at − 70 °C, the activities of caspase-3, − 8 and − 9 in samples were measured by colorimetric assay with a plate reader.
Statistical analysis
The statistically significant differences between PEDV infected cells and controls in the induction of apoptosis and caspase activation and, between the recombinants plasmid transfected cells and pEGFP-N1 transfected control in the induction of apoptosis was determined by unpaired T-tests or ANOVA accordingly with GraphPad Prism (Version 5.0) software. Differences were considered statistically at a value of p < 0.05.
Discussion
In this study, we observed that PEDV SM98 and CV777 strains induce apoptosis in Vero, Vero-E6 and Marc-145 cells with distinct cytopathic effects (CPEs) including roundup, cell fusion, vacuolation, syncytium formation etc. Membrane blebbing and the translocation of phosphatidi lserine to the cell surface, as well as nuclear concentration and fragmentation, were all observed in PEDV infected cells.
Mechanically, PEDV SM98 and CV777 virus infection induced a significant cleavage on AIFM1 suggested an involvement of mitochondrial-mediated apoptosis pathway, which is consistent with previous observation [
19]. Besides this pathway, we found the extrinsic apoptotic pathway was also activated by SM98 and CV777 infection as caspase-8, caspase3 and PARP were all cleaved in response to PEDV virus infection. This study enriched the knowledge of the PEDV infection induced apoptosis through a complicated pattern, apart from the mitochondrial-mediated apoptosis pathway, the extrinsic apoptotic pathway was also activated, no matter whether or not it is original. Consistently, many viruses from coronavirus family induced apoptosis with caspase activation: such as TGEV could cause infected ST cells apoptosis in a caspase-dependent way, accompanied by DNA cleavage and mitochondrial transmembrane potential change [
21]; IBV could cause a caspase-dependent apoptosis, as characterized by chromosomal condensation, DNA fragmentation, caspase-3 activation, and poly (ADP-ribose) polymerase degradation [
22]; CCoV, is responsible for enteric disease (diarrhea, vomiting, dehydration, loss of appetite and occasional death) in young puppies [
23,
24], could cause infected cells apoptosis with activation caspase-3 [
23]; SARS-CoV infection could results in the downregulation of Bcl-2, the activation of caspase-3, as well as the upregulation of Bax, suggesting the involvement of the caspase family and the activation of the mitochondrial signaling pathway [
25]. MERS-CoV could efficiently infect human primary T lymphocytes and activates the extrinsic and intrinsic apoptosis pathway [
26]. However, the exact mechanism especially the critical viral inducer of PEDV has not yet illustrated.
To date, PEDV has evolved into two distinct clades, the classical strains and high-virulent field strains [
14]. Antigenic variations of PEDV spike protein (S) between these two groups were thought to contribute to the severity of recent outbreaks [
27,
28], as the S protein plays a pivotal role in cell adsorption, membrane fusion, and induction of neutralizing antibodies [
29,
30]. Here we demonstrate that S1 protein indeed is a predominant apoptosis inducer (while S2 is not) among all the structural and nonstructural proteins of PEDV, although M and Nsp1, 2, 5, 6, 7, 9 and 13 also induce cell apoptosis but to a less extent. Furthermore, the S1 protein of circulating variant strain PEDV BJ2011 also induce significant apoptosis in host cells, and the ability to induce apoptosis in Vero-E6 is greater than that of the cell adapted strain, such as CV777. This suggested S1 protein is a critical protein mediated PEDV-induced cell apoptosis and contributes partly to its virulence, which is consistent with our laboratory’s recent report [
31]. Among all the identified proteins capable inducing apoptosis in host cells, the M protein is the most abundant component of the viral envelope and plays a central role in virus morphogenesis and assembly via its interactions with other viral proteins [
32]. The pro-apoptotic role of the SARS-CoV M protein has been revealed by disrupting the interaction of PDK1 with PKB/Akt[
33,
34]. This suggests that PEDV M protein may also activate apoptosis through this pathway. 3C-like protease (3CLpro) has been demonstrated could cause cell growth arrest and apoptosis in SARS-CoV 3CLpro-expressing human promonocyte cells [
35]. Similarly, the pro-apoptotic function of PEDV Nsp5 i.e. 3C-like protease also has observed in this study. Coronavirus Nsp1 is the first N-terminal cleavage product of the pp1a and pp1a/b polyproteins [
36]. Coronavirus Nsp1 regulates host cell and virus gene expression [
37] that functions as a potent IFN antagonist. PEDV Nsp1 has been demonstrated could block the nuclear translocation of IRF1 and reduce the number of peroxisomes to suppress IRF1-mediated type III IFNs activities [
38]. Here we identified a novel function of PEDV Nsp1 as an inducer of apoptosis.
Coronaviruses can infect a wide range of mammals and birds, but exhibit a marked tropism for epithelial cells of the respiratory and enteric tracts, as well as for macrophages [
16,
39], and most of them can induce apoptosis in infected host cells. Although a number of cellular mechanisms and gene products (such as the SARS-CoV M, N protein[
34,
40] and 3C-like protease (3CLpro) [
35]) capable of inducing apoptosis have been revealed, the S1 protein of these coronaviruses have not yet well-evaluated. Although the amino acid sequence of these S proteins are divergent greatly (Additional file
1: Table S1), here we demonstrated that S1 proteins of all these coronaviruses tested in this study were able to induce apoptosis in transfected Vero-E6 cells. Given the fact that a number of coronaviruses gene products could induce apoptosis in host cells, it is proposed that the spike protein S1 may function as a cofactor that could enhance or stimulate extrinsic apoptotic pathway in these coronavirus induced apoptosis.