Avian encephalomyelitis virus nonstructural protein 2C induces apoptosis by activating cytochrome c/caspase-9 pathway.

The nonstructural protein 2C is highly conserved among picornaviruses and plays an important role in the assembly of mature virions, membrane association, and viral RNA synthesis. The investigation of other potential functions of nonstructural protein 2C from avian encephalomyelitis virus (AEV) resulted in identifying for the first time that the protein 2C is involved in apoptosis. Expression of the protein 2C on chick embryo brain (CEB) and Cos-7 cells produced TUNEL-positive cells characterized by a cleavage of cellular DNA and the formation of membrane-enclosed apoptotic bodies. Analysis of the protein 2C showed that the N-terminal domain containing 35 amino acid (aa) residues (between 46 and 80 aa) is associated with apoptotic function. Transfection of the deletion mutant lacking this 35 aa's into CEB and Cos-7 cells failed to induce apoptosis. Furthermore, the protein 2C induced apoptosis in the transfected CEB and Cos-7 cells through activation of caspase-9 rather than caspase-8 followed by activation of caspase-3 pathway. Analysis of the Western blots of caspase-3 and caspase-9 showed the characteristics of active caspase-3 and -9 in the 2C-transfected CEB and Cos-7 cells as seen in the AEV-infected CEB cells while they were in the form of procaspase-3 and procaspase-9 in the 2C mutant-transfected cells. To further elucidate the mechanism of the 2C-induced apoptosis, the 2C-transfected CEB and Cos-7 cells were fractionated into mitochondria and cytosol and subjected for Western blotting, located cytochrome c in the mitochondria as well as the cytosol fractions, while it was only sequestered in the mitochondrial fraction in the mutant 2C-transfected cells. The protein 2C was located in the mitochondria and cytosol of the transfected/infected CEB and transfected Cos-7 cells, but the mutant lost its ability to localize to the mitochondria. Altogether, the results demonstrate that the protein 2C localized to the mitochondria of the transfected cells triggered the efflux of cytochrome c into the cytosol in turn activating the upstream caspase-9 and then the downstream caspase-3, thus leading to apoptosis in the cells.

Pathogenesis of avian encephalomyelitis viruses.

The pathogenesis of a field strain, a vaccine strain and the egg-adapted Van Roekel strain of avian encephalomyelitis virus in susceptible chicken embryos and day-old chickens was investigated using enzyme-linked immunosorbent assays for the detection of virus-specific antibody and antigen. The Van Roekel strain was shown to be highly neurotropic whereas the field and vaccine strains were enterotropic. Radioimmuno-precipitation studies using Na125I-labelled purified virus failed to detect any differences in the composition of the structural viral proteins of each strain that could account for these differences. As expected, the field and vaccine strains were more efficient than the Van Roekel strain at inducing antibody following oral administration. Primary cultures of chicken embryo brain cells supported the growth of the Van Roekel strain to a much greater extent than the field and vaccine strains.

Comparative pathogenicity of avian encephalomyelitis viruses in chicken embryos.

Multiplications of wild, various embryo-adapting and completely embryo-adapted avian encephalomyelitis (AE) viruses in chicken embryos were compared by the fluorescent-antibody technique (FAT). With a wild AE virus, viral antigens were randomly seen in the central nervous system (CNS), appearing least often in the cerebellum. Other organs seldom became test positive, except for heart and kidney. Even with 4 chicken brain-passaged viruses in the process of embryo adaptation, there was little augmentation of antigens except in the alimentary tract. However, the 2 midpassage viruses showed a peculiar localization of antigens in the white matter of the lumbosacral cord, together with the appearance of test-positive spinal ganglion cells. With 2 strains of embryo-adapted AE virus, the antigens appeared first in the spinal ganglion cells and secondly in the lumbosacral cord and then spread to the cerebrum. Subsequently, clinical signs of AE were evident. This peculiar invasion order was a prominent feature.