| Apoptosis is a normal physiological process involved in cellular and developmental immunity. Upon receiving an apoptotic signal a group of cysteine proteases called caspases are activated. The first caspases activated are the upstream initiator caspases such as Drosophila DRONC, which then cleave and activate the downstream effector caspases. Those effector caspases including Drosophila DrICE, then selectively cleave various cellular substrates leading to the eventual dismantling of the cell. In the case of virus infection, this apoptotic response by the host cell can result in premature cell death, thereby limiting the spread and/or pathogenesis of infection in the organism. Therefore, many viruses have evolved strategies to combat this process and thereby allow for their replication.; The work presented here is divided into two sections examining the regulation of apoptosis in dipteran and in lepidopteran insect cells infected with entomopoxvirus. The first section examines the role of mitochondrial factors such as cytochrome c in caspase activation. Although mitochondrial proteins play well-defined roles in caspase activation in mammalian cells, the role of mitochondrial factors in caspase activation in insects is not as clear. Using cell-free extracts, we provide evidence that mitochondrial factors are neither required for, nor involved in, Drosophila caspase activation. Cytosolic extract from apoptotic S2 cells induced caspase activation when mixed with cytosolic extract from normal S2 cells, even when caspases were inhibited in the apoptotic extract. These results indicate that caspase activation in Drosophila is regulated solely by cytoplasmic factors.; The second section describes the characterization of a gene identified from Amsacta moorei entomopoxvirus (AmEPV) with low but significant homology to anti-apoptotic baculovirus p35 genes. This gene is predicted to encode a protein of 32.7 kd with putative anti-apoptotic function. In keeping with previous nomenclature we have renamed the gene AMVp33 . When ectopically expressed, AMVp33 blocked apoptosis in Spodoptera frugiperda-derived Sf21 cells, Drosophila melanogaster-derived S2 cells, Lymantria dispar-derived LD652Y cells and human fibrosarcoma HT-1080 cells. P33 was a stoichiometric inhibitor of DrICE, DCP-1, and Sf-caspase-1, but not DRONC. P33 was cleaved by DrICE and the cleavage fragments stably associated with DrICE. Thus, AMVp33 encodes a substrate inhibitor similar to P35. |
