Stress-Induced Apoptosis in Spodoptera frugiperda (Sf9) Cells: Baculovirus p35 Mitigates eIF2α Phosphorylation

Abstract

Spodopterafrugiperda (SJ9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2α phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2α phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcδ35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2α phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2α, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2α partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2α phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2α phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2α phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2α phosphorylation.