Porcine epidemic diarrhea virus S1 protein is the critical inducer of apoptosis

Abstract

Background: Porcine Epidemic Diarrhea (PED) is an acute and highly contagious enteric disease caused by PED virus (PEDV), characterized by vomitting, watery diarrhea and fatal dehydration with high mortality in sucking piglets of one week of age. Although PEDV induced cell apoptosis has been established in vitro and in vivo, the functional protein that contributes to this event remains unclear.

Methods: The activation or cleavage of main apoptosis-associated molecular such as AIFM1, caspase-3, caspase-8, caspase-9 and PARP in PEDV infected host cells were analyzed by western blotting. The nuclear change of infected cell was monitored by confocal immunofluorescence assay. The overexpressing plasmids of 16 non-structural proteins (Nsp1-16) and 6 structural proteins (M, N, E, ORF3, S1 and S2) were constructed by cloning. Cell apoptosis induced by PEDV or overexpression non-structural or structural proteins was measured by the flow cytometry assay.

Results: PEDV could infect various host cells including Vero, Vero-E6 and Marc-145 and cause obvious cytopathic effects, including roundup, cell fusion, cell membrane vacuolation, syncytium formation and cause apparent apoptosis. In infected cells, PEDV-induced apoptosis is accompanied by nuclear concentration and fragmentation as a result of caspase-3 and caspase-8 activation and AIFM1 and PARP cleavage. Overexpression of S1 Spike protein of PEDV SM98 strain effectively induced host cell apoptosis, while the expression of the other non-structure proteins (Nsp1-16) and structural proteins (M, N, E, S2 and ORF3) has no or less effect on cell apoptosis. Similarly, expression of S1 protein from wild-type strain BJ2011 or cell-adapted strain CV777, also induce apoptosis in transfected cells. Finally, we demonstrated that the S1 proteins from various coronavirus family members such as TGEV, IBV, CCoV, SARS and MERS could also induce Vero-E6 cells apoptosis.

Conclusion: S1 Spike protein is one of the most critical functional proteins that contribute to cell apoptosis. Expression of S1 proteins of the coronavirus tested in this study could all induce cell apoptosis suggesting S1 maybe is an effective inducer in Coronavirus-induced cell apoptosis and targeting S1 protein expression probably is a promising strategy to inhibit coronavirus infection and thus mediated apoptosis on host cells.

Keywords: Apoptosis; Apoptosis-inducing factor mitochondria associated 1 (AIFM1); Porcine epidemic diarrhea virus (PEDV); Spike S1 protein.

Fig. 1

PEDV infection results in obvious cytopathic effects.(a, b, c) Vero-E6, Vero and Marc-145 cells mock control. (d, e, f) CPE of Vero-E6, Vero and Marc-145 cells infected with SM98. (g, h, i) CPE of Vero-E6, Vero and Mark-145 cells infected with CV777

Fig. 2

PEDV infection results in Apparent Apoptosis in Vero, Vero-E6 and MARC145 cells. Mock and infected Vero, Vero-E6 and MARC145 cells were collected and stained with 7-AAD and annexin-V-PE at 48 h post infection, and then analyzed by flow cytometry. (a) PEDV SM98 and CV777 strains both could induce significant apoptosis in Vero-E6, Vero and MARC-145 cells. (b, d, f) Percentages of annexin-V-PE positive cells from SM98 and CV777 infected Vero-E6, Vero and Marc-145 cells. (c, e, g) Percentages of annexin-V-PE positive and 7AAD negative cells from SM98 and CV777 infected Vero-E6, Vero and Marc-145 cells. Results are representative of three independent experiments. Data are represented as mean ± SD, n = 3. (**, p < 0.01; ***, p < 0.001;ns, no significant).

Fig. 3

PEDV infection results in cell nuclear DAN fragmentation and chromatin condensation. Vero cells grown on coverslips were infected with PEDV SM98 at a multiplicity of infection of 0.1, The cells were fixed and incubated with mice anti PEDV S monoclonal antibody and DAPI at 12, 48 and 72 h post infection. (fluoview 1000×)

Fig. 4

PEDV infection activates AIFM1, Caspase-3 and Caspase-8. Mock and infected Vero cells were collected at 12, 24, 36, 48, 60 and 72 h post infection and split with RIPA lysis buffer and supersonic. (a-d) Cleaved AIFM1, Caspase-3, Caspase-8 and PARP were analyzed by western blotting. (e-j) The graphs show the quantification of WB bands in panel A-D by testing optical density using ImageJ - system, normalized to β-actin and expressed relative to mock infection. Two biological replicated experiments were performed for each protein, and one result was represented

Fig. 5

PEDV infection results in the activation of Caspase-3 and Caspase-8,but not Caspase-9. The activation of Caspase-3, Caspase-8 and Caspase-9 were detected by the Caspase activity assay kits. The relative fold change (OD405nm) of Caspase-3, − 8 and − 9 activities are shown with graphs. Data are represented as mean + /− SD, n = 3.(***, p < 0.001;ns,no significant)

Fig. 6

PEDV S1 Protein is the Critical inducer of apoptosis among all the Structural and Nonstructural Proteins. pEGFP-N1 and all the recombinant plasmids were transfected into confluent Vero E6 monolayer cells respectively. 48 h later, the cells were harvested and analyzed by Fluorescence-activated cells sorter (FACS), GFP-positive cells were gated for apoptosis analysis. (a)The results showed that PEDV S1 Protein is the Critical inducer of Apoptosis in host Cells. (b) Percentages of annexin-V-PE positive cells from gated cells. (c) Percentages of annexin-V-PE and 7AAD double positive cells from gated cells. Results are representative of three independent experiments. Results are representative of three independent experiments. Data are represented as mean + /− SD, n = 3. (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

Fig. 7

PEDV CV777 and new-emerging strain BJ2011 S1 protein Could also induce apoptosis in VERO-E6 even in BHK-21 cells. pEGFP-N1 and the recombinant plasmids expressing CV777 or BJ2011 S1 protein were transfected into confluent Vero-E6 or BHK-21 monolayer cells respectively. 48 h later, the cells were harvested and analyzed by Fluorescence-activated cells sorter (FACS), GFP-positive cells were gated for apoptosis analysis. (a) PEDV CV777 and new-emerging strain BJ2011 S1 protein Could also induce apoptosis in Vero-E6 even in BHK-21 cells. (b) Percentages of annexin-V-PE positive Vero-E6 Cells from gated cells are shown with graphs. Results are representative of three independent experiments. (c) Percentages of annexin-V-PE positive BHK-21 Cells from gated cells are shown with graphs. Results are representative of three independent experiments. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significant)

Fig. 8

TGEV, IBV, CoCoV, SARS and MERS CoV Spike Protein S1 Could also Induce Apoptosis in Vero-E6 cells. TGEV, IBV, CCoV, SARS and MERS S1 genes were cloned or synthesized in fusion with EGFP gene and were transfected into VERO-E6, 48 h later, the cells were harvested and analyzed by Fluorescence-activated cells sorter (FACS), GFP-positive cells were gated for apoptosis analysis. (a) TGEV, IBV, CCoV, SARS and MERS S1 genes were correctly constructed in fusion with EGFP. The empty vector pEGFP-N1 and the recombinant plasmids were all transfected into confluent BHK-21 monolayer cells separately. 24 h later, bright green fluorescence was observed from the all the recombinant plasmids transfected cells. (b) The results showed that TGEV, IBV, CCoV, SARS and MERS S1 Protein Could Induce Apoptosis in Vero-E6 cells. (c)Percentages of annexin-V-PE positive cells from gated cells are shown with graphs. Results are representative of three independent experiments. (*, p < 0.05; **, p < 0.01; ***, p < 0.001)