Simian Varicella Virus Induces Apoptosis in Monkey Kidney Cells by the Intrinsic Pathway and Involves Downregulation of Bcl-2 Expression

Simian varicella virus (SVV) causes varicella in primates, becomes latent in ganglionic neurons, and reactivates to produce zoster. SVV produces a cytopathic effect in monkey kidney cells in tissue culture. To study the mechanism by which SVV-infected cells die, we examined markers of apoptosis 24 to 64 h postinfection (hpi). Western blot analysis of virus-infected cell lysates revealed a significant increase in the levels of the cleaved active form of caspase-3, accompanied by a parallel increase in caspase-3 activity at 40 to 64 hpi. Caspase-9, a marker for the intrinsic pathway, was activated significantly in SVV-infected cells at all time points, whereas trace levels of the active form of caspase-8, an extrinsic pathway marker, was detected only at 64 hpi. Bcl-2 expression at the mRNA and protein levels was decreased by 50 to 70% throughout the course of virus infection. Release of cytochrome c, an activator of caspase-9, from mitochondria into the cytoplasm was increased by 200% at 64 hpi. Analysis of Vero cells infected with SVV expressing green fluorescent protein (SVV-GFP) at 64 hpi revealed colocalization of the active forms of caspase-3 and caspase-9 and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining with GFP. A significant decrease in the bcl-2 mRNA levels along with an abundance of mRNA specific for SVV genes 63, 40, and 21 was seen in the fraction of Vero cells that were infected with SVV-GFP. Together, these findings indicate that SVV induces apoptosis in cultured Vero cells through the intrinsic pathway in which Bcl-2 is downregulated.Apoptosis, a regulated form of cell death, plays a critical role in the homeostasis of multicellular organisms. Key features include membrane blebbing, chromatin condensation, and cell shrinkage. UV irradiation, deprivation of growth factors, and viral infection all cause apoptosis in cultured cells. Apoptosis is triggered by sequential activation of a group of cysteine proteases known as caspases. Apoptosis proceeds primarily through two pathways. The extrinsic pathway involves activation of caspase-8 and is initiated by ligand interaction with Fas or death receptors, while the intrinsic pathway is activated by an imbalance between proapoptotic (e.g., Bad and Bax) and antiapoptotic (e.g., Bcl-2 and Bcl-xL) proteins in mitochondria (21), resulting in release of cytochrome c from mitochondria, which in turn activates caspase-9. Bcl-2 plays an important role in cell survival (22, 32). Both caspase-8 and caspase-9 activate caspase-3, which along with other effector caspases, cleave critical cellular proteins, resulting in apoptosis.Simian varicella virus (SVV), the primate counterpart of human varicella zoster virus (VZV), produces a naturally occurring exanthematous disease that mimics human varicella (9, 18). Clinical and pathological changes produced by SVV infection of primates are similar to those produced by human varicella, and both VZV and SVV reactivate from latently infected ganglionic neurons (4, 13, 23, 33). The SVV and VZV genomes share a high degree of nucleotide homology (3, 10), and SVV-specific antibodies cross-react with human VZV in serum neutralization and complement fixation tests (5, 6, 30). Both viruses produce a cytopathic effect in monkey kidney cells in tissue culture (2, 29, 31). VZV has been shown to cause apoptosis in cultured Vero cells, human foreskin fibroblasts, and peripheral blood mononuclear cells isolated from healthy donors but not in primary human dorsal root ganglionic neurons (12, 13, 16, 28). Apoptosis is also seen in peripheral blood mononuclear cells of children infected with VZV in vivo (25). Thus, VZV-induced apoptosis may be cell type specific. The main objectives of this study were to determine if SVV induces apoptosis in cultured Vero cells, a monkey kidney cell line, and to identify the specific pathways.