Porcine sapelovirus 2A protein induces mitochondrial-dependent apoptosis

Figures

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  Figure 1. PSV infection induces apoptosis. (A) The cells infected with PSV (MOI=1) for 24 h were stained with DAPI, and then viewed under fluorescence microscope. (B, C) PSV-infected cells with different MOI (0.5, 1, and 2) were collected and subjected to dual Annexin V and propidium iodide (PI) labelling, and then detected by flow cytometry. The graph represented the percentage of Annexin V positive quadrants (n=3). *P < 0.05, **P < 0.01. (D) Mock and PSV-infected cells (MOI=1) were fixed at 24 hpi, sequentially, labelled with TUNEL (green), anti-PSV-VP1 antibody (red), and DAPI. The TUNEL labelling and VP1 protein staining cells were observed using fluorescence microscope. Scale bar: 100 μm.

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  Figure 2. PSV infection induces caspase activation and PARP1 cleavage. (A, B) PSV-infected cells with different MOI (0.5, 1, and 2) were collected at 24 hpi. The activation of caspase-8, -9, -3 and the cleavage of PARP1 were analyzed by Western blot. GAPDH was used as internal loading control.

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  Figure 3. Caspase inhibitors affect PSV-induced apoptosis. (A) ST cells were pretreated with caspase inhibitors (Z-VAD-FMK, Z-LEHD-FMK, and Z-IEHD-FMK) (20 or 30 μM) and infected with PSV. After 24 h, the cellular lysates were detected by Western blot using antibodies against PSV-VP1 and PARP1. (B, C) ST cells were pretreated with caspase inhibitors at 30 μM, followed by PSV infection. Cells were harvested at 24 hpi, stained with Annexin V and PI, and then examined through flow cytometry. The graph represented the percentage of Annexin V positive quadrants (n = 3). **P < 0.01.

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  Figure 4. Caspase inhibitors affect PSV infection. (A) ST cells were treated with Z-VAD-FMK and infected with PSV. After 24 h, cells were fixed and incubated with anti-PSV-VP1 antibody and DAPI, followed by examined using fluorescence microscope. Scale bar: 100 μm. (B) After 36 h, the viral titers were determined by the Spearman-Kärber method, and three independent experiments were expressed as the mean ± SD. *P < 0.05. (C-F) The PSV replication in the presence of Z-LEHD-FMK and Z-IEHD-FMK. *P < 0.05; ns, no significant difference.

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  Figure 5. Identification of PSV-encoded proteins that induce apoptosis. (A) The expression plasmids of all PSV-encoded proteins were constructed and respectively transfected into HEK293A for 24 h. Then the cellular lysates were detected by Western blot with antibodies against Flag and PARP1 to detect the expression levels of viral proteins and the PARP1 cleavage. The asteroids indicate VP4, 2B protein bands. (B, C) The HEK293A cells were transfected with the expression plasmids of all PSV-encoded proteins, respectively. After 24 h, the cells were harvested and subjected to dual Annexin V and PI labelling, sequentially, detected by flow cytometry. The graph represented the percentage of Annexin V positive quadrants (n = 3). *P < 0.05, **P < 0.01.

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  Figure 6. PSV 2A induces apoptosis. (A) The PSV 2A-transfected cells were fixed and incubated with TUNEL (green), anti-Flag antibody (red), and DAPI at 24 h, and then observed under fluorescence microscope. Scale bar: 100 μm. (B, C) The 2A-transfected cells were stained with Annexin V/PI, and detected by flow cytometry analysis. The graph represented the percentage of Annexin V positive quadrants (n = 3). *P < 0.05, **P < 0.01. (D) The activation of caspase-8, -9, -3 were analyzed via Western blot, after pCAGGS-Flag-2A transfected for 24 h. (E, F) The activation of Bax and Bcl-2 in 2A-transfected cells were detected at 24 h by Western blot. Ratios of the gray density of Bax and Bcl-2 were analyzed by Image J software. Densitometric data for Bax/Bcl-2 from three independent experiments are expressed as the mean ± SD. **P < 0.01.

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  Figure 7. PSV 2A induces cytochrome C release and loss of mitochondrial membrane potential. (A) The cytosolic and mitochondrial fractions of PSV 2A-transfected HEK293A cells were isolated, and then the cytochrome C (cyto C) in cytosolic and mitochondrial fractions were analyzed by Western blot. (B, C) For detecting the mitochondrial membrane potential, the HEK293A cells were transfected with pCAGGS-Flag-2A for 24 h, the cells were labelled with JC-1 and detected through flow cytometry. The graph represents the JC-1 fluorescence ratio (Red/Green). *P < 0.05.

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  Figure 8. The crucial residues of PSV 2A for inducing the apoptosis. (A) The amino acid alignments of 2A in representative members of picornaviruses were listed. Identical residues are indicated by dots on black background. The residues marked with red arrow represent the amino acids that form the catalytic triad of poliovirus 2A. The residues marked with blue arrow are other amino acids selected as control. Residues are numbered according to the amino acid sequence of EV71 MH685713, CVB3 MF678329, EV-C NP_740477.1, EV-D68 KX351830, BEV NC_001859, RVA-89 NC_001617, and RV-B06 JX193795. (B, C) The HEK293A cells were transfected with pCAGGS-Flag-2A and its mutants: pCAGGS-Flag-2A(H48A), pCAGGS-Flag-2A(H58A), pCAGGS-Flag-2A(D91A), pCAGGS-Flag-2A(H125A), and pCAGGS-Flag-2A(C164A), after 24 h, the cells were collected and subjected to dual Annexin V and PI labelling, sequentially, detected by flow cytometry. The graph represented the percentage of Annexin V positive quadrants (n = 3). **P < 0.01. (D) HEK293A cells were transfected with pCAGGS-Flag-2A and its mutants for 24 h. Then the cellular lysates were analyzed through western blot with antibodies against Flag and PARP1.