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Comparative Study
. 2001 Jul;75(14):6402-9.
doi: 10.1128/JVI.75.14.6402-6409.2001.

Induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus

C Liu  1 , H Y Xu, D X Liu
Affiliations
Comparative Study

Induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus

C Liu et al. J Virol. 2001 Jul.

Abstract

Avian coronavirus infectious bronchitis virus (IBV) is the causative agent of chicken infectious bronchitis, an acute, highly contagious viral respiratory disease. Replication of IBV in Vero cells causes extensive cytopathic effects (CPE), leading to destruction of the entire monolayer and the death of infected cells. In this study, we investigated the cell death processes during acute IBV infection and the underlying mechanisms. The results show that both necrosis and apoptosis may contribute to the death of infected cells in lytic IBV infection. Caspase-dependent apoptosis, as characterized by chromosomal condensation, DNA fragmentation, caspase-3 activation, and poly(ADP-ribose) polymerase degradation, was detected in IBV-infected Vero cells. Addition of the general caspase inhibitor z-VAD-FMK to the culture media showed inhibition of the hallmarks of apoptosis and increase of the release of virus to the culture media at 16 h postinfection. However, neither the necrotic process nor the productive replication of IBV in Vero cells was severely affected by the inhibition of apoptosis. Screening of 11 IBV-encoded proteins suggested that a 58-kDa mature cleavage product could induce apoptotic changes in cells transiently expressing the protein. This study adds one more example to the growing list of animal viruses that induce apoptosis during their replication cycles.

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Figures

FIG. 1
FIG. 1
Morphological changes in IBV-infected Vero cells. Cells were mock (M) or IBV (I) infected, stained with Hoechst 33342 at 16 and 36 h postinfection (pi), and viewed in a light microscope. Phase, phase-contrast images; Hoechst, nuclear staining.
FIG. 2
FIG. 2
(a) DNA fragmentation assay of IBV-infected Vero cells. Low-molecular-weight DNA was isolated from mock (M)- (lanes 2 to 4) and IBV (I)-infected Vero cells harvested at 8 (lane 5), 24 (lane 6), and 48 (lane 7) h postinfection (pi) and analyzed on a 2% agarose gel. The 1-kb ladder DNA markers (lane 1) were purchased from Gibco BRL (Life Technologies). (b) TUNEL assay of mock- and IBV-infected cells at 8, 24, and 48 h postinfection.
FIG. 3
FIG. 3
(a) Effects of z-VAD-FMK on DNA fragmentation in IBV-infected Vero cells. Cells were infected with 2 PFU of IBV per cell in the presence of DMSO (20 μl/ml) (lanes 4 and 5) or z-VAD-FMK (20 μg/ml) (lanes 2 and 3). Low-molecular-weight DNA was isolated from IBV-infected (I) Vero cells at 16 (lanes 2 and 4) and 36 (lanes 3 and 5) h postinfection and analyzed on a 2% agarose gel. The 1-kb ladder DNA markers (lane 1) were purchased from Gibco BRL (Life Technologies). (b) Effects of z-VAD-FMK on morphological changes of nuclei of IBV-infected Vero cells. Cells were infected with 2 PFU of IBV per cell in the presence of DMSO (20 μl/ml) or z-VAD-FMK (20 μg/ml). The nuclei were stained with Hochest 33342 at 36 h postinfection and viewed with a light microscope.
FIG. 4
FIG. 4
(a) Western blotting analysis of caspase-3 in IBV-infected Vero cells. Cells were mock (M) or IBV (I) infected in the presence of DMSO (20 μl/ml) (lanes 1 to 3) or z-VAD-FMK (20 μg/ml) (lanes 4 to 6) and harvested at 24 and 48 h postinfection (pi). Caspase-3 was analyzed by separation of total proteins on SDS–17.5% polyacrylamide gels, transfer to a nitrocellular membrane, and blotting with a rabbit anti-caspase-3 polyclonal antibody (PharMingen). The protein was detected by using an ECL+Plus Western blotting detection kit (Amersham Pharmacia Biotech). Numbers on the left indicate molecular masses in kilodaltons. (b) Western blotting analysis of PARP in IBV-infected Vero cells. Cells were mock or IBV infected in the presence of DMSO (20 μl/ml) (lanes 1 to 3) or z-VAD-FMK (20 μg/ml) (lanes 4 to 6) and harvested at 24 and 48 h postinfection. PARP was analyzed by separation of total proteins on SDS–7.5% polyacrylamide gels, transfer to a nitrocellular membrane, and blotting with an anti-PARP 3 monoclonal antibody (PharMingen).
FIG. 5
FIG. 5
(a) Western blotting analysis of the E and 58-kDa proteins overexpressed in BHK cells in a Sindbis virus expression system. Cells were transfected with RNA transcribed from plasmids as indicated above each lane by electroporation and were harvested at 48 h posttransfection. Gel electrophoresis of the total proteins was performed on SDS–17.5% (lanes 1 to 3) and 10% (lanes 4 to 6) polyacrylamide gels, respectively. The proteins were transferred to nitrocellular membranes, blotted with rabbit anti-E and anti-58-kDa protein polyclonal antibodies, respectively, and detected by using an ECL+Plus Western blotting detection kit (Amersham Pharmacia Biotech). Numbers on the left indicate molecular masses in kilodaltons. (b) Induction of DNA fragmentation in BHK cells by overexpression of the 58-kDa protein in a Sindbis virus expression system. Cells were transfected with RNA transcribed from plasmids as indicated above each lane by electroporation. Low-molecular-weight DNA was isolated at 48 h posttransfection and analyzed on a 2% agarose gel. The 1-kb ladder DNA markers (lane 1) were purchased from Gibco BRL (Life Technologies).
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