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.

FIG. 1.

(A) TMEV final gene products arranged on the polyprotein in a standard picornavirus format of the L protein: four P1 capsid proteins and three P2 and four P3 nonstructural proteins. The 18-kDa out-of-frame L* protein, which is initiated 13 nucleotides downstream of the authentic polyprotein AUG, is shown below the polyprotein. (B) All of the BeAn virus nonstructural proteins except the 20-amino-acid 3B (VPg) were assembled using overlap extension PCR into the pIRES vector downstream of the BeAn virus IRES so that each protein is translated from a 5′ AUG codon and contains a C-terminal Flag sequence. The L protein was also assembled into the same vector without an IRES for cap-dependent translation.

FIG. 2.

SDS-PAGE analysis of in vitro-translated RNAs transcribed from pIRES-3C, -3D, and -3CD plasmids in RRL in the presence of [³⁵S]methionine. Translation of pIRES-3C^pro reveals no apparent labeling of 3C^pro (lane 1); pIRES-3D^pol shows labeling of the 53-kDa 3D^pro (lane 2); pIRES-3CD shows labeling of the 65-kDa 3CD (lane 3); and when pIRES 3CD is overexposed, labeling of 3CD, 3D^pro, and 23-kDa 3C^pro is shown (lane −4).

FIG. 3.

SDS-PAGE of the expression of pIRES-BeAn virus nonstructural gene constructs in transiently transfected BHK-12 cells. (A) Immunoblot analysis at 24 h (even lanes) and 48 h (odd lanes) posttransfection. Lanes: 1, empty vector; 2 and 3, L; 4 and 5, 2A; 6 and 7, 2B; 8 and 9, 2C; 10 and 11, 3A; 12 and 13, 3D polymerase. (B) Time course of the expression of 3C^pro in the presence of 20 μM of the reversible 26S proteasome inhibitor MG132. MW, molecular weight (in thousands).

FIG. 4.

Cytotoxicity determined by the WST-1 assay at 24 and 48 h after transfection of plasmid BeAn virus nonstructural gene constructs in BHK-21 cells. *, P < 0.05; **, P < 0.01. (A) Cell death after IRES-dependent expression of virus nonstructural genes (mean ± standard error; n = 3 to 6). (B) Cell death after cap-dependent expression of L (mean ± standard error; n = 3). (C) Comparison of cytotoxicity after cap-dependent expression of L and the L CHCC mutant (mean ± standard error; n = 3). (D) Time course of cap-dependent expression of L up to 20 h posttransfection.

FIG. 5.

Digital confocal microscopic images of nuclei of cells loaded with the marker protein GFP (GFP_NLS) (A to C) and Flag-tagged BeAn virus L (D and E) in BHK-21 cells 24 h posttransfection. Cells transfected with pL showed efflux of GFP from the nucleus to the cytoplasm (A), whereas cells transfected with pEV (B) and the pL mutant plasmid DNA (C) retained GFP in the nucleus. Indirect immunofluorescent antibody staining of L in the cytoplasm of BHK-21 cells 24 h after pL transfection showed DAPI-stained nuclei (D) and L stained with rabbit anti-Flag M2 and anti-rabbit IgG-Alexa Fluor 568 and nuclei stained with DAPI (E). Bars = 10 μm.

FIG. 6.

Effect of pL and the pL Zn finger mutant in transfected BHK-21 cells. (A) FACS analysis of PI- and annexin V-stained cells at 24 h reveals increased numbers of apoptotic cells (right lower quadrant) after pL transfection but not after transfection with the pL mutant. (B) Immunoblots revealing PARP cleavage and caspase 3 and caspase 9 cleavage to their active forms, p17 and p37, respectively, but no caspase 8 cleavage to the active p18 form. (C) Effect of treatment of pL-transfected cells with the pancaspase inhibitor qVD-OPh, showing a significant reduction (P < 0.01) in cell death compared to treatment with PBS at 24 and 48 h. (D) Immunoblot of pL-transfected cells treated with qVD-OPh, revealing a lack of PARP and caspase 3 cleavages at 20 to 24 h. (E) Electron photomicrographs of pL (top)- and pEV (bottom)-transfected cells at 24 h showing an apoptotic cell with condensed nuclear chromatin, two cells in the early stages of apoptosis, one with loss of marginated nuclear chromatin, and necrotic cell fragments. Cells in the lower frame appear normal except for ruffling of the cell membrane. The photomicrographs are at the same magnification. Bar = 5 μm.

FIG. 7.

Bak and Bax expression in BHK-21 cells transfected with pL. The immunoblot reveals increased Bak expression at 12 to 24 h but decreased Bax over the same period.

FIG. 8.

Effect of pL and the pL Zn finger mutant in transfected M1-D macrophages. (A) FACS analysis of PI- and annexin V-stained cells at 24 h revealed increased numbers of apoptotic cells (right lower quadrant) after pL transfection but not after transfection of the pL mutant. (B) Immunoblot revealing PARP cleavage at 24 h.