Generally, cell death occurs by necrosis and apoptosis. Necrosis is the result of immediate, physically damage to cells causing swelling, rupture and release of cellular contents. Apoptosis, on the other hand, is a regulated cascade of physiological response culminating in cell death. Apoptosis is recognized by cell shrinkage followed by plasma membrane blebbing and release of apoptotic bodies. In vivo, the apoptotic bodies undergo phagocytosis by surrounding cells. In culture, where phagocytosis is limited, secondary breakdown of apoptotic bodies occurs releasing cellular constituents into the medium.
Although the apoptotic program is highly complex and the cellular events involve the activation of many signaling cascades, two main pathways predominate; the mitochondrial-mediated pathway and the cell surface-mediated signal transduction pathway. The mitochondria, which are the energy generators for the cell, also house apoptosis-instigating molecules including cytochrome c, Smac/DIABLO, and AIF (5, 10, 16). These molecules are released in response to apoptotic stimuli such as toxins or DNA damage and promote activation of pro-caspase 9, a principal cysteine-aspartate protease responsible for initiating cellular apoptosis signaling. Caspase 9 as well as other caspases exist in an inactive zymogen pro-caspase state. Proteolytic processing is required to convert the pro-caspase into a proteolytically active caspase molecule. The cell surface-mediated signal transduction pathway is triggered through the binding of ligands to death receptors at the cell plasma membrane, allowing for the recruitment of deathincluding complexes at the cytoplasmic tail of the receptors. The classic model for this process involves binding of the trimeric Fas ligand to the Fas receptor. A conformational change ensues allowing for the recruitment of the proteins FADD and pro-caspase 8 to the cytoplasmic death domain of the receptor. This complex formation leads to caspase 8 activation. The mitochondrial-mediated and cell surface signal transduction pathways converge at the activation of caspase 3. Thus, at the apex of the two apoptosis cascades are the initiator caspases, caspase 8 on the cell surface signaling pathway and caspase 9 in the mitochondrial-mediated pathway. The initiator caspases 8 and 9 in turn activate the downstream effector class of caspases 3, 6 and 7. The activation of these effector caspases leads to the final execution of the cell death program.
Viruses have evolved novel mechanisms to prevent apoptosis of the infected host cell and thereby promote virus multiplication (12, 13). The baculovirus Autographa californica nucleopolyhedrovirus (Ac MNPV) was the first insect virus known to induce and inhibit apoptosis (3). Infection of Spodopera frugiperda Sf-21 cells with the wide-type (wt) AcMNPV results in the production of both budded virus and occluded virus. In contrast, infection with an AcMNPV mutant lacking the P35 gene results in induction of apoptosis with limited budded virus production and no occluded virus production (14). To date, two distinct types of apoptotic suppressors, represented by P35 and the inhibitors of apoptosis (IAPs), have been identified in the invertebrate baculoviruses (2). Baculovirus IAPs were the first members of the IAP protein family to be discovered (4, 11). The best-studied viral IAP, that from the Orgyia pseudotsugata MNPV (OpMNPV), prevents apoptosis in insects and mammals by a mechanism that includes interaction with itself and pro-apoptotic proteins like Reaper, HID and GRIM of Drosophila (1, 7, 9, 17, 18)
SeMNPV has been registered as a biological insecticide to control the target pest(19). Its genome contains two iap genes (20), the IAPs interactaction with Ubiquitin has been reported (22) but their function in vivo is unkown. In this study, we cloned and expressed the SeMNPV iap3 gene in HEK293 cells and demonstrated the anti-apoptotic activity of SeMNPV IAP3 in the mammalian HEK293 cells.
The Function of SeMNPV IAP3 in Mammalian Cells
- Received Date: 25 May 2007
- Accepted Date: 04 January 2008
Abstract: The baculoviral inhibitors of apoptosis play a significant role in infectivity and viral host-range, which make them potential candidates for the engineering and improvement of baculovirus insecticidal. The iap3 gene of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), amplified by PCR, was 939 bp encoding IAP3. The PCR product was cloned into EcoR I /Bam H I of the plasmid pEGFP-C1. GFP was fused to the N-terminaus of IAP3 to study distribution in HEK293. It was observed that the plasmid expressing IAP3 significantly inhibited apoptosis induced by cisplatin in HEK293 cells. We conclude that the IAP3 of SeMNPV is functional in mammalian cells.