Induction of apoptosis by sindbis virus occurs at cell entry and does not require virus replication

Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and ca n lead to the apoptotic death of infected cells. To determine the step in v irus replication during which apoptosis is triggered, we used UV-inactivate d SV, chemicals that block virus fusion or protein synthesis, and cells tha t do and do not express heparan sulfate, the initial binding molecule for S V infection of many cells. In initial experiments, UV-inactivated neuroadap ted SV (NSV) induced apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate in the presence of cycloheximide. When fusion of prebound UV-inactivated NSV was rapidly induced at the plasma membrane by exposure t o acidic pH, apoptosis was induced in CHO cells with or without heparan sul fate in the presence or absence of cycloheximide in a virus dose-dependent manner. In N18 neuroblastoma cells, the relative virulence of the virus str ain was an important determinant of apoptosis induced by UV-inactivated SV. Treatment of N18 cells with monensin to prevent endosomal acidification an hour before, but not 2 h after, exposure to live NSV blocked the induction of cell death, as did treatment with NH4Cl or bafilomycin A1. These studie s indicate that SV can induce apoptosis at the time of fusion with the cell membrane and that virus replication is not required.