Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein alpha as an apoptosis inducer

Abstract

Betanodaviruses, members of the Nodaviridae family, are the causative agents of viral nervous necrosis in fish and infection by which cause high mortality in larvae and juveniles in a wide range of marine fish species in Asia, Europe, Australia, Martinique, and Tahit. Greasy grouper (Epinephelus tauvina) nervous necrosis viruses (GGNNV) were investigated for their apoptotic activity in culture cells. GGNNV infection of sea bass (SB) cells appeared to induce a typical cytopathic effect (CPE), i.e., cytoplasmic vacuolation, thinning, rounding up, detachment of infected cells from the cultured dish, and eventually cell lysis and death. The infected SB cells underwent DNA fragmentation and stained positive in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay, suggesting that GGNNV infection induced apoptosis in SB cells. In addition, GGNNV-infected SB cells showed an increased activity of caspase-8-like proteases (IETDase) and caspase-3-like proteases (IETDase), whereas inhibitor of caspase-8 and caspase-3 reduced GGNNV-induced apoptosis. This suggests that GGNNV may promote apoptosis via the extrinsic pathway in SB cells. Protein alpha, the precursor of GGNNV capsid proteins, was transiently expressed in SB and Cos-7 cells. The DNA fragmentation and TUNEL positive signal were apparent in SB and Cos-7 cells expressing protein alpha, suggesting that protein alpha may serve as an apoptotic inducer in these cells. Moreover, expression of protein alpha resulted in the activation of caspase-3-like proteases in both cells, which could be inhibited by a caspase-3-like protease specific inhibitor DEVD-CHO peptide. These results suggest that fish caspases are important elements in GGNNV-meditated apoptosis.

Fig. 1

(a) Expression of GGNNV proteins A and α in prokaryotic expression system. Total cell lysates induced by IPTG showing the expression of recombinant protein A (lane 2) and α (lane 4) were electrophoresed in a 12% SDS-PAGE and stained with Coomassie brilliant blue. Lanes 1 and 3 showed noninduced control. M indicates the molecular mass in kilodaltons. (b) Immunoblotting assay of GGNNV proteins A and α. Total cell lysates of GGNNV-infected SB cells were transferred to nitrocellulose membrane and detected by the mono-specific antibodies against proteins A (lane 1) and α (lane 2), respectively. Control sera (lane 3) and lysates from uninfected SB cells (lane 4) were used as negative control. M indicates the molecular mass in kilodaltons.

Fig. 2

CPE and nuclear morphology of GGNNV-infected SB cells. GGNNV infected (D) and mock-infected SB cells (B) were stained with Hoechst 33342 at 48 h post-infection to visualize nuclear morphology. C shows the CPE at 48 h PI. A refers to mock-infected cells at 48 h. Magnification, X200.

Fig. 3

(a) Time course study of betanodavirus GGNNV-induced fragmentation of cellular DNA in SB cells. DNA was extracted from GGNNV-infected SB cells and electrophoresed on 2% agarose gel. Lanes 3 to 7 indicated DNA extracted from cells at 24, 36, 48, 60, and 72 h PI, respectively. Lane 2 refers to mock-infected cells at 60 h PI. Lane 1 contains the 100 bp DNA size marker. (b) TUNEL assay showing apoptosis in GGNNV infected SB cells. TUNEL assay was performed at 24 h (B), 36 h (C), and 48 h (D and E) PI. Mock-infected cells (A) were checked at 48 h PI. Green indicates TUNEL positive cells. Image E shows that SB cells were treated with DEVD-CHO. Cells were observed under a confocal microscope and photographed in 250X magnification.

Fig. 4

Betanodavirus GGNNV infection induced activation of caspase-8-like proteases in SB cells. Cell lysates were harvested at 24 and 48 h PI and assayed for IETDase activity using caspase-8 colorimetric substrate IETD-pNA. Simultaneously, Mock-infected SB cells were used as a negative control. Values shown are means from duplicate experiments.

Fig. 5

Betanodavirus GGNNV infection induced activation of caspase-3-like proteases in SB cells. Cell lysates were harvested at 24 and 48 h PI and assayed for EDVDase activity using caspase-3 colorimetric substrate DEVD-pNA. Simultaneously, Mock-infected SB cells were used as a negative control. Values shown are means from duplicate experiments.

Fig. 6

Immunoblotting assay showing the expression of proteins A and α in eukaryotic expression system. Cell lysates from Cos-7 and SB cells transfected with pEGFP-RNA1 and pEGFP-RNA2 were electrophoresed in a 12% SDS-PAGE, transferred to nitrocellulose membrane, and detected by the monospecific antibodies against proteins A (lanes 1 and 4) and α (lanes 2 and 5), respectively. Lysates from cells transfected with pEGFP-C1 (lanes 3 and 6) were indicated as negative control.

Fig. 7

(a) Cellular DNA fragmentation induced by transiently expressing protein α in SB and Cos-7 cells. DNA samples were extracted from cells transfected with pEGFP-RNA2 (lane 2) pEGFP-RNA1 (lane 3) and pEGFP-C1 (lane 4) at 48 h post-transfection and DNA fragmentation were detected by 2.0% agarose gel electrophoresis. Lane 1 indicates the DNA size marker. (b) Detection of apoptosis in SB and Cos-7 by TUNEL-labeling. SB or Cos-7 cells were transfected with plasmid pEGFP-RNA2. At 48 h post-transfection, the cells were fixed and analyzed for detection of apoptosis by TUNEL staining, which detects the DNA breakage (brown color). A and C indicate TUNEL-labeling of Cos-7 and SB cells that were transfected with EGFP-RNA2 at 48 h post-transfection, respectively. B and D indicate Cos-7 and SB cells that were transfected with pEGFP-C1 at 48 h post-transfection, respectively. Magnification X200.

Fig. 8

Protein α induces activation of caspase-3 proteases in both SB and Cos-7 cells. Cell lysates from SB or Cos-7 cells transfected with plasmids pEGFP-RNA2 or pEGFP-C1 were harvested at 24 h post-transfection and assayed for DEVDase activity using the caspase-3 colorimetric substrate DEVD-pNA. In addition, cells transfected with pEGFP-RNA2 were treated with DEVD-CHO. The pEGFP-C1 was used as a negative control and to maintain equal plasmid DNA concentration for each of the transfections. Values shown are means from duplicate experiments.