AMV063 of Betaentomopoxvirus amoorei encodes a functional Caspase-2 essential for virus-mediated apoptosis

The genome of Betaentomopoxvirus amoorei (AMEV) is approximately 232 kb in size and encodes around 256 predicted open reading frames (ORFs), many of which are associated with host interaction and apoptosis modulation. A comprehensive functional characterization of the AMV063 ORF was conducted in this study. The AMV063 protein was heterologously expressed using a baculovirus-based expression system. In parallel, gene silencing of the AMV063 ORF was achieved by deleting the corresponding sequence and generating a recombinant AmΔ063/gfp virus through homologous recombination. To investigate functional outcomes, apoptosis-sensitive CF-70-B2 cells were either treated with purified AMV063 protein, infected with the recombinant AmΔ063/gfp virus, or both. After treatment, the caspase-2 enzymatic activity was measured, apoptotic responses were assessed using TUNEL staining, and cell viability was measured via the trypan blue exclusion test. Exposure to AMV063 protein resulted in a marked increase in caspase-2 activity, and a corresponding elevation in TUNEL-positive cell death. Conversely, in cells infected with the AmΔ063/gfp virus, caspase-2 activity, TUNEL positivity, and overall cell death were significantly reduced compared with those infected with the control AmΔsph/gfp virus. These findings indicate that the AMV063 ORF sequence encodes a protein associated with caspase-2-like activity and that the recombinant AMV063 protein is capable of inducing apoptotic responses in host cells. This study represents the first functional characterization of the AMV063 ORF, demonstrating that it is a functional caspase-2-like gene.