Expression of murine leukemia virus envelope protein is sufficient for the induction of apoptosis

Abstract

The generation of cytopathic effects by murine leukemia viruses (MLVs) in different cell types correlates with the ability of the virus to induce thymic lymphoma. We showed that the induction of apoptosis in mink epithelial cells by mink cell focus-forming (MCF) MLV infection results in the accumulation of high levels of both unintegrated viral DNA and the envelope precursor polyprotein (gPr80(env)). Comparisons of envelope protein expression levels of plasmid clones of the env gene of the MCF13 and noncytopathic NZB-9 MLV strains demonstrated that the accumulation of MCF13 gPr80(env) results in endoplasmic reticulum stress and is sufficient for the induction of apoptosis.

FIG. 1.

Analysis of MCF13 and NZB-9 MLV envelope proteins. (A) Western blot of protein extracts prepared at 24, 48, and 72 h after transfection of 1.6 × 10⁶ mink epithelial cells with 25 μl Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and 25 μg purified plasmid DNA of either empty pLNCX2 vector (P) or clones of MCF13 env (M) or NZB-9 env (N). The faint band detectable in the control (P) lanes resulted from nonspecific binding of primary MAb 83A25 (9). Protein extracts from mink cells productively infected with the MCF13 (C1) or NZB-9 (C2) virus were used as controls for migration of the envelope precursor (gPr80^env) and SU for each MLV, which are indicated with arrows. α-tubulin was detected as a loading control. (B) Pulse-chase analysis of envelope proteins in mink cells infected with MCF13 or NZB-9 MLV at a multiplicity of infection of 2.5 for 4 days. The cells were pulse-labeled for 30 min with [³⁵S]Met-Cys and chased for various times. For each time point, 200 μg of protein extract was reacted with polyclonal goat anti-Rauscher murine leukemia virus gp70 serum kindly provided by S. Ruscetti (NCI, Frederick, MD). The immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lane labeled “serum” shows an immunoprecipitation control performed with normal goat serum.

FIG. 2.

Killing of mink epithelial cells by Env. Mink cells were transfected as described for Fig. 1 with plasmid DNA corresponding to either empty pLNCX2 vector, MCF13 env, or NZB-9 env. Forty-eight hours later, the cells were trypsinized, plated at dilutions of 20- and 40-fold onto two 10-cm plates for each dilution, and grown in selectable medium containing 1,200 μg per ml of G418 for 15 to 19 days. Neomycin-resistant (neo^R) colonies were counted after being stained with crystal blue dye. The mean values and standard deviations of the numbers of colonies for mink cells transfected with the empty vector (black bar), MCF13 env (white bar), or NZB-9 env (striped bar) were calculated from the results of two independent experiments.

FIG. 3.

MCF13 Env expression induces apoptosis in transfected cells. Mink epithelial cells were transfected as described for Fig. 1. Adherent and nonadherent cells were pooled from a 10-cm plate at 48 h after transfection, fixed with 1% paraformaldehyde and 0.1% Triton X-100 for 10 min, and stained with a solution of 1 μg per ml of Hoechst 33342 dye (Molecular Probes, Eugene, OR) for 5 min. Nuclei were visualized with a Zeiss Axiophot fluorescence microscope and a Plan-NEOFLUAR 20X/0.5 objective utilizing a UV filter. Percentages of apoptotic cells are shown at the indicated times after transfection with plasmids containing either empty pLNCX2 vector (black bars), MCF13 env (white bars), or NZB-9 env (striped bars). Mean values and standard deviations were calculated from examining 380 to 1,200 transfected cells in each of two independent experiments.

FIG. 4.

MCF13 Env upregulates CHOP and GRP78 in transfected cells. Mink epithelial cells were transfected as described for Fig. 1. Western blotting was performed with antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) to (A) CHOP or (B) GRP78 on protein extracts that were prepared at 24, 48, and 72 h after transfection with empty pLNCX2 vector (P), MCF13 env (M), or NZB-9 env (N). Cell extracts from mink cells either untreated (Un) or treated with 1 μg per ml of tunicamycin for 18 h (Tm) were used as controls for ER stress. Increases (n-fold [fold-increase]) in intensity compared with that of the band in the respective control (P) lane, which was given the arbitrary value of 1, are indicated for the CHOP and GRP78 bands detectable for MCF13 (M) and NZB-9 (N). α-tubulin was detected as a loading control.