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

Abstract

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 Ͻ1% 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...

Apoptotic and Antiapoptotic Activity of L Protein of Theiler's Murine Encephalomyelitis Virus

“…Fan et al (9) recently reported that transfection of an expression construct of BeAn L into BHK-21 cells and a mouse macrophage cell line led to cell death and apoptosis. Romanova et al (18) found evidence of antiapoptotic activity of EMCV and mengovirus L in infections by these cardioviruses in HeLa cells.…”

“…A previous study found that apoptosis triggered by transfection in BHK-21 cells of expression constructs of BeAn L required p53 (9). In order to further investigate this issue, we infected HCT116 colorectal cancer cells that constitutively express p53 or do not express p53.…”

“…1), in regulating apoptosis. In vitro studies of TMEV carried out by Fan et al (9) showed that transfection of an expression construct of BeAn L into BHK-21 cells and a mouse macrophage cell line led to cell death and apoptosis, while Romanova et al (18) found that L of other cardioviruses has antiapoptotic activity, since infection with a mengovirus with a mutation in the L zinc-binding domain led to apoptosis of HeLa cells that was not seen following wild-type (wt) mengovirus infection. In order to clarify the latter observations and further characterize the apoptotic activity of TMEV, we investigated apoptosis in different cell types following transfection of DA and GDVII L expression constructs and following infection with DA and GDVII wt and L mutant viruses.…”

Cultivation and Serological Characterization of a Human Theiler's-Like Cardiovirus Associated with Diarrheal Disease

“…The marked difference in CPE between HTCV-UC6 and SAFV-3 (NL2007) may also reflect sequence divergence in the L protein (Fig. 6), which in TMEV has been found to mediate apoptosis in infected cells (8). Furthermore, even in the most susceptible lines, individual cells may differ in their permissivity for viral replication due to factors such as inherent heterogeneity of the cell line or cell culture density.…”

Role(s) of Leader protein of Saffold virus

“…9 TMEV-L is a multifunctional protein that plays important roles in neurovirulence, anti-interferon (IFN) activity and viral persistence. 8 Furthermore, TMEV-L has a pro-apoptotic activity; 10,11 although, in part, it also has antiapoptotic activity depending on the TMEV strain and the particular cell types, 12 such as encephalomyocarditis virus (EMCV)-L. 13 From these observations, TMEV-L should be a key factor in inducing the pathological changes found after infection of the mouse. TMEV-L has a zinc finger domain in the N-terminus, acidic domain in the middle region and S/T-rich domain in the C-terminus.…”