Semum forest virus induced programmed cell death

Semliki Forest virus (SFV) is a small positive stranded RNA virus that infects both neurons and myelin forming glial cells of the mouse nervous system. The replication of the A7(74) strain of SFV is complete and productive in embryonic and neonatal mouse neurons, but is restricted in neurons of adult mice. This is associated with a change from widespread to focal infection of the CNS and determines death or survival for infected individuals. Previous studies on SFV and the related alphavirus, sindbis, have shown that cells activate a program of death by suicide following infection. To elucidate how SFV infection induces cell death we characterised the mechanism of death using low density primary cultures prepared from the CNS & PNS of mice and a number of continuous cell lines. Cell death in the cell lines was by suicide as determined by light and electron microscopy and TUNEL staining. Virusinduced death of cultured neurons appeared similar to the cell death induced by trophic factor deprivation and also possessed the morphological hallmarks of apoptosis. Overexpression in a number of cell Unes of the anti-apoptotic gene bcl-2 suppressed cell death, affected virus yield and gave rise to a persistent infection. Infection of immature neurons in vivo also resulted in death by apoptosis. The ICE family of novel cysteine proteases is pivotal in the implementation of cell suicide. We show that proteolysis of the specific CPP32/apopain substrate, poly ADPribose polymerase (PARP), to the 89 kilodalton fragment begins prior to the formation of a significant viral cytopathic effect (12 hours). PARP cleavage was maximal by 72 hours postinfection of BHK cells with both virulent and avirulent strains of SFV. Overexpression of bcl-2 in the AT-3 cell line completely abrogated the formation of the proteolytic fragment following infection. Methylketone and aldehyde derivatives of the tetrapeptide ICE family inhibitors, zVAD and DEVD, partially inhibited the programmed cell death of NGF-dependent mouse sensory neurons induced by either SFV infection or NGF deprivation. PARP cleavage occured in cerebellar granule neurons dying by apoptosis following SFV infection in vitro. Analysing the fundamental neurobiology of virus and host cell interactions is essential to our understanding of how CNS viral infections cause acute or chronic neurological disorders including viral encephalitides.