Neuronal cell death is induced by the Ni-CE (CE) rabies fixed strain, but not its progenitor, the Nishigahara (Ni) strain. This CE-strain cytopathic effect (CPE) reportedly occurs due to amino acid substitution at position 95 in the matrix protein (M95). A Ni mutant with a valine-to-alanine modification at M95 [Ni(M95A)] induced cell death whereas a CE mutant with the reverse modification [CE(M95V)] did not. This phenomenon of M95-related cell death resembles apoptosis but has not been fully examined. Therefore, we aimed to elucidate its mechanism in human neuroblastoma SK-N-SH cells infected with the original and modified CE and Ni strains. Cells infected with each strain showed high surface exposure of phosphatidylserine, an early apoptosis signal. However, only CE-infected and Ni(M95A)-infected cells showed membrane disruption. We thus speculated that the M95 mutation is involved in cell membrane disruption in late apoptosis, and subjected SK-N-SH cells infected with each strain to apoptosis-executioner caspase-3/7 activity assays for further investigation. We found no significant difference in caspase-3/7 activity between rabies-strain infected cells and mock-infected cells, suggesting that the M95 mutation is not implicated in late apoptosis. We then investigated rabies-strain CPEs and cell viability in the presence of the caspase inhibitor Z-VAD-FMK. CPE was greater and viability was lower in CE-infected and Ni(M95A)-infected cells than Ni-infected and CE(M95V)-infected cells, regardless of the presence or absence of Z-VAD-FMK. Therefore, our results suggest that rabies virus M95-related cell death is caspase-independent and not apoptotic.
