High-neurovirulence GDVII virus induces apoptosis in murine astrocytes through tumor necrosis factor (TNF)-receptor and TNF-related apoptosis-inducing ligand

Abstract

We carried out a study to determine if the high-neurovirulence GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) and the demyelinating, low-neurovirulence BeAn strain induced apoptosis in murine astrocytes. Astrocytes, the major glial cell population of the central nervous system, were semipermissive for GDVII virus replication. Programmed cell death, demonstrated by apoptosis-specific caspase-3 protease activity, was maximal 8 h after GDVII infection at an m.o.i. of 1. Purified TMEV capsid proteins VP1, VP2, and VP3 did not induce apoptosis but antibodies to VP1 and VP2 inhibited it. Antibody inhibition of caspase-3 activity as well as flow cytometry experiments implicated TNF-related apoptosis-inducing ligand (TRAIL) and TNF-alpha-receptor (TNF-R) in apoptosis signaling. Conversely, TNF-alpha and the TRAIL-receptor were not upregulated. Furthermore, the number of functional TNF-alpha receptors, but not their affinity, was increased in apoptotic GDVII virus-infected astrocytes, as confirmed in binding experiments with 125I-labeled recombinant murine TNF-alpha. In vivo studies showed that most of the cells loaded with the virus when injected in the brains of SJL mice were neurons but very few showed TUNEL costaining. Conversely, many of the apoptotic cells found were also positive for GFAP staining.

Fig. 1

Semilog graph showing virus production in supernatants of BeAn- and GDVII virus-infected astrocytes measured by plaque assay on BHK-21 cell monolayers. Cells (3 × 10³) were infected at m.o.i.s of 1, 10, and 100 for 45 min at room temperature. Residual virus remaining from the inoculum were washed three times and cultures were replenished with complete medium. The supernatants were centrifuged and tested 24 h postinfection. Circles (•, ○) indicate titers detected at a m.o.i. of 100 just after the washes were completed and numbers in parentheses indicate PFU/cell.

Fig. 2

Caspase-3 activity in GDVII-infected astrocyte cultures. Astrocytes were mock-infected (0), or infected at m.o.i.s of 1, 10, or 100 for 24 h. Cells infected at an m.o.i. of 10 were simultaneously treated with the irreversible pan-caspase inhibitor Z-VAD-FMK at a final concentration of 50 μM (Z-VAD). Cell extracts were tested for caspase-3 activity according to assay conditions of the CaspACE kit. Each enzymatic reaction contained 50 μg of total protein of cell extracts. A positive control was obtained by treating cultures with 200 nM Staurosporine (+). Error bars indicated SD of three independent experiments. *, indicated significant differences calculated by Student’s test, P < 0.05.

Fig. 3

Kinetics of caspase-3 induction in astrocyte cultures infected at an m.o.i. of 10 with GDVII virus for 0 to 48 h.

Fig. 4

Inhibition of the GDVII virus-induced apoptotic cell death by anti-TMEV antiserum. Purified virus was preincubated for 30 min at 37°C with indicated dilutions of rabbit antiserum or normal rabbit serum (NRS) diluted 10⁻² and added to the astrocytic cultures. Caspase-3 activity was analyzed 24 h postinfection. Results represent mean values ± SD of triplicate samples.

Fig. 5

Apoptosis in astrocytes cultures to which TMEV VP1, VP2, or VP3 native proteins, purified by high-pressure liquid chromatography, were absorbed. Monolayer cells were treated three-fold with the amount of pure proteins, equivalent to an m.o.i. of 10, which was also used for infection with intact GDVII and BeAn virions.

Fig. 6

Expression of cytoplasmic TRAIL protein and cell surface TNF-R in astrocytes as determined by flow cytometry. Cells were mock-infected or infected with the nonneurovirulent strain BeAn (shaded profiles), or infected at an m.o.i. of 10 with GDVII virus for 8 h (thick lines). Stainings obtained with normal goat IgG or rabbit serum instead of primary antibodies are shown (thin lines) as negative controls. The font sizes are different in the two panels (80 and 100 counts).

Fig. 7

Scatchard analysis of specific ¹²⁵I-TNF-α binding to mock-infected or GDVII virus-infected astrocytes. The ratio bound/free against bound ¹²⁵I-TNF-α was plotted. The linear plot indicates a simple bimolecular reaction with a single type of receptor.

Fig. 8

Inhibition of GDVII-induced cell death by anti-TRAIL and anti-TNF-R antibodies. Cells were infected, washed, and cultured for 24 h in the presence of either increasing concentrations of affinity-purified antibody or decreasing dilutions of antiserum. Caspase-3 activity was further determined as described under Materials and Methods. Negative controls were provided by normal rabbit serum (diluted 10⁻²) and normal purified goat IgG (10 μg/ml) (−). Results are given as mean percentage inhibition ± SD of infected cells based on absorbance at 405 nm in triplicate samples.

Fig. 9

Confocal images of astrocyte cultures infected for 24 h at an m.o.i. of 10 with GDVII. Staining for GFAP (A), TUNEL (B), or merged images (C) were shown. Scale bar, 30 μm.

Fig. 10

Sections of brain stained for GFAP (A), TUNEL (B and E), or replicating TMEV (D) at the level of the nucleus accumbens region 4 days after intracerebral inoculation of virus. In C and F, merged images of respective rows shows colocalization of staining in some cells. Scale bar in C and F, 30 μm.