Theiler's Murine Encephalomyelitis Virus Leader Protein Is the Only Nonstructural Protein Tested That Induces Apoptosis When Transfected into Mammalian Cells

Theiler''s murine encephalomyelitis virus (TMEV) induces two distinct cell death programs, necrosis and apoptosis. The apoptotic pathway is of particular interest because TMEV persists in the central nervous system of mice, largely in infiltrating macrophages, which undergo apoptosis. Infection of murine macrophages in culture induces apoptosis that is Bax dependent through the intrinsic or mitochondrial pathway, restricting infectious-virus yields and raising the possibility that apoptosis represents a mechanism to attenuate TMEV yet promote macrophage-to-macrophage spread during persistent infection. To help define the cellular stressors and upstream signaling events leading to apoptosis during TMEV infection, we screened baby hamster kidney (BHK-21) cells transfected to express individual nonstructural genes (except 3B) of the low-neurovirulence BeAn virus strain for cell death. Only expression of the leader protein led to apoptosis, as assessed by fluorescence-activated cell sorting analysis of propidium iodide- and annexin V-stained transfected cells, immunoblot analysis of poly(ADP-ribose) polymerase and caspase cleavages, electron microscopy, and inhibition of apoptosis by the pancaspase inhibitor qVD-OPh. After transfection, Bak and not Bax expression increased, suggesting that the apical pathway leading to activation of these Bcl-2 multi-BH-domain proapoptotic proteins differs in BeAn virus infection versus L transfection. Mutation to remove the CHCC Zn finger motif from L, a motif required by L to mediate inhibition of nucleocytoplasmic trafficking, significantly reduced L-protein-induced apoptosis in both BHK-21 and M1-D macrophages.Theiler''s murine encephalomyelitis viruses (TMEV), members of the genus Cardiovirus in the family Picornaviridae, are highly cytolytic RNA viruses. Mice experimentally infected with a low-neurovirulence TMEV, such as BeAn virus, develop persistent infection in the central nervous system (CNS) and an inflammatory demyelinating disease, providing an experimental analogue for multiple sclerosis. BeAn virus persists primarily in macrophages in the CNS of infected mice. Schlitt et al. (34) found that 74% of TUNEL-positive cells in infected spinal cords (primarily in CNS lesions) were T and B lymphocytes and 8% were macrophages, although virus genomes were detected in <1% of apoptotic cells, consistent with infection of only a low percentage of macrophages and the fact that TMEV does not infect T or B lymphocytes in culture. Thus, some means other than direct infection was responsible for apoptosis of most CNS macrophages, including TMEV triggering apoptosis through tumor necrosis factor alpha or tumor necrosis factor alpha-related apoptosis-inducing ligand by binding death receptors on activated macrophages in vitro, as reported elsewhere (17).Infection of mouse macrophages induces apoptosis (16, 26) mediated by Bax through the intrinsic or mitochondrial pathway and severely restricts the yield of progeny virus (37). Thus, apoptosis may be a mechanism to attenuate the virus yet promote macrophage-to-macrophage spread through phagocytosis of infected apoptotic blebs during persistence (37). In contrast, TMEV infection in other rodent cells tested thus far, including baby hamster kidney (BHK-21) cells, produces necrotic cell death with high virus yields. The contrasting outcomes of TMEV infection point to the existence of two distinct virus-induced cell death programs.The genes of an increasing number of RNA viruses have been shown to encode proteins that trigger apoptosis. Among picornaviruses, coxsackievirus B3 1B (VP2) (12, 13), avian encephalomyocarditis virus 1C (VP3) (24) and 2C (25), enterovirus 71 2A (20), and poliovirus 2A (10) and 3C protease (3C^pro) (3) induce apoptosis, mostly through the intrinsic pathway. Coxsackievirus B3 VP2 has been shown to interact with the proapoptotic Siva protein in a yeast two-hybrid screen (12), but exactly how the VP2-Siva interaction or any of the other picornavirus proteins initiates the apoptotic cascade remains unknown.To gain insight into the upstream signaling events that lead to apoptosis, we tested the ability of individual BeAn virus nonstructural genes to induce apoptosis in uninfected BHK-21 cells. Only the leader (L) protein resulted in apoptosis and mutation of the CHCC Zn finger motif in L significantly reduced L protein-induced apoptosis.