Regulation of Theiler’s Virus Induced Cell Death

Theiler’s murine encephalomyelitis virus (TMEV) is a highly cytolytic RNA virus that causes a persistent central nervous system infection and immune-mediated demyelination in susceptible strains of mice. TMEV-infected macrophages, undergo apoptosis and restrict virus replication (<10 pfu/cell). In contrast, TMEV infection of other rodent cells tested, e.g., baby hamster kidney cells (BHK-21), produces necrotic cell death with high virus yields (200-500 pfu/cell). The contrasting outcomes of TMEV infection alone suggest the existence of distinct virus-induced cell death pathways. We found that Mcl-1 was the only Bcl-2 prosurvival protein that differed substantially in expression in different cell lines: Mcl-1 was highly expressed in BHK-21 cells but expressed at low levels in murine macrophage cell lines. FACS analysis showed that 50% of BeAn virus-infected BHK-21 cells that were transfected with Mcl-1 siRNA died an apoptotic cell death while only ~14% of infected parental BHK-21 cells died by apoptosis (the rest of cells died by necrosis). Similar results were observed by constitutive Mcl-1 siRNA expression in BHK-21 cells and knock-down also resulted in a 100-fold reduction in viral titers compared to parental BHK-21 cells. As a result, Mcl-1 protects against apoptosis in BHK-21 cells, which shifts the balance of cell death from apoptosis to necrosis. Temporal analysis of TMEV infection in macrophages with infection in BHK-21 cells for comparison, showed no appreciable change in viral RNA replication, polyprotein processing or assembly of protomers and pentamers. However, we found that mature 160S virions contained incompletely cleaved VP0 (to VP4 and VP2) and a faster migrating 216S peak that contained minor amounts of novel capsid fragments of 21- and 17-kDa in immunoblots with polyclonal rabbit antisera to BeAn virus. The faster migrating peak predominated at later times post-infection and had ~100-fold higher particle to pfu ratios than in the 160S virion peak, indicating that these virions were defective. BeAn virions incubated with recombinant caspase 3 in vitro revealed the novel capsid fragments as well as loss of VP4, suggesting a global conformational change at late times during one-step viral kinetics.