Functional analysis of putative amsacta moorei entomopoxvirus (AMEV) caspase orf (AMV063)

Amsacta moorei entomopoxvirus (AMEV) infects agriculturally important pests. Genetic engineering studies on the genome of AMEV helped to reveal undiscovered gene functions and also its potential to be used as biotechnological agent. It has been shown that AMEV infects and causes apoptosis of insect cells, such as Ld652 and CF-70-B2. Caspases are members of cysteine protease family and have important roles in apoptosis. Although the interaction of AMEV putative caspase ORF (AMV063) with protein kinase of AMEV has been determined, the apoptosis and caspase activity of these proteins has not been investigated. In this thesis, functional analysis of AMEV063 ORF was performed. AMV063 protein was expressed in the prokaryotic system. Knock-out mutant, Am∆063/gfp was created by deleting its sequence with homologue recombination. Apoptosis and caspase-2 activities of apoptosis sensitive CF-70-B2 cells were investigated by treating AMV063 protein and Am∆063/gfp mutant with different combinations. Caspase-2 and TUNEL activity could not be determined in AMV063 protein the treated cells. Significant decrease in caspase-2 and TUNEL activity was observed in the cells treated with Am∆063/gfp compared to the same cell line treated with Am∆sph/gfp. These results clearly indicate that AMV063 sequence encodes the caspase-2 protein and only AMV063 protein produced in its natural system has caspase-2 and apoptosis activity. Keywords: Insect viruses, Amsacta moorei entomopoxvirus, AMV063, Am∆063/gfp, caspase-2, apoptosis