Induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of E protein as an apoptosis inducer

Abstract

We demonstrated that infection of 17Cl-1 cells with the murine coronavirus mouse hepatitis virus (MHV) induced caspase-dependent apoptosis. MHV-infected DBT cells did not show apoptotic changes, indicating that apoptosis was not a universal mechanism of cell death in MHV-infected cells. Expression of MHV structural proteins by recombinant vaccinia viruses showed that expression of MHV E protein induced apoptosis in DBT cells, whereas expression of other MHV structural proteins, including S protein, M protein, N protein, and hemagglutinin-esterase protein, failed to induce apoptosis. MHV E protein-mediated apoptosis was suppressed by a high level of Bcl-2 oncogene expression. Our data showed that MHV E protein is a multifunctional protein; in addition to its known function in coronavirus envelope formation, it also induces apoptosis.

FIG. 1

DNA fragmentation analysis of 17Cl-1 cells infected with MHV. Low-molecular-weight DNA was isolated at 50 and 70 h p.i. from MHV-A59-infected, MHV-JHM-infected, or mock-infected cells, as indicated. Samples were electrophoresed in a 2% agarose gel and visualized with ethidium bromide.

FIG. 2

DNA fragmentation in MHV-infected 17Cl-1 cells. 17Cl-1 cells were mock infected (A) or infected with MHV-A59 at an MOI of 5 (B). At 36 h p.i., cells attached to the plates were examined by TUNEL assay for DNA fragmentation.

FIG. 3

Chromatin condensation in 17Cl-1 cells infected with MHV. 17Cl-1 cells were mock infected (A) or infected with MHV-A59 at an MOI of 5 (B). At 68 h p.i., mock-infected cells (A) and floating cells in MHV-infected culture media (B) were collected and stained with Hoechst 33342.

FIG. 4

Effect of Z-DEVD-fmk on MHV-induced apoptosis. 17Cl-1 cells were mock infected or incubated with medium containing Z-DEVD-fmk (dissolved in dimethyl sulfoxide) at a concentration of 0 (dimethyl sulfoxide only), 40, or 80 μM, as indicated, before and after infection with MHV-A59. At 48 h p.i., low-molecular-weight DNA was extracted and separated in a 2% agarose gel.

FIG. 5

Induction of apoptosis in VV-E-infected DBT cells. DBT cells were mock infected or infected with VV-IHD or VV-E at an MOI of 1, as indicated. At 45 h p.i., low-molecular-weight DNA was extracted and separated in a 2% agarose gel.

FIG. 6

Chromatin condensation in DBT cells infected with VV-E. DBT cells were mock infected (A) or infected with wt vaccinia virus (B) or VV-E (C) at an MOI of 1. At 42 h p.i., the cells were collected and stained with Hoechst 33342.

FIG. 7

Effect of high-level Bcl-2 expression on E protein-induced apoptosis. (A) Expression of Bcl-2 protein in DBT, DBT/neo, and DBT/bcl-2 cells. Proteins were separated, membranes were probed, and the signal was visualized as described in the text. (B) Inhibition of E protein-induced apoptosis in DBT/bcl-2 cells. DBT, DBT/neo, and DBT/bcl-2 cells were infected with VV-E at an MOI of 1 or mock infected, as indicated. At 48 h p.i., low-molecular-weight DNA was extracted and separated in a 2% agarose gel.