The pathogenicity of some retroviruses correlates with their ability to induce cytopathic effects involving different cell types (1
). Diseases induced by cytopathic retroviruses include malignancies, immunodeficiency, and neurodegeneration. An example of a cytopathic murine leukemia virus (MLV) that induces thymic T-cell lymphoma is the mink cell focus-forming (MCF) MLV (5
). Upon characterizing the early events that occur during the development of thymic lymphoma induced by inoculation of the MCF13 MLV strain into AKR mice, we detected a significant reduction of thymic lymphocytes via apoptosis (36
). To better understand this phenomenon, we established an in vitro culture system utilizing CCL64 mink epithelial cells, which similarly undergo apoptosis after infection with MCF13 MLV (18
It has been demonstrated for several cytopathic retroviruses that there is a strong correlation between virus superinfection and cell killing (4
). One of the results of retroviral superinfection is the accumulation of high levels of unintegrated viral DNA in cells, which has been implicated in the induction of cytopathic effects (4
). An additional consequence of superinfection by some pathogenic retroviruses, including MCF13 MLV, is the accumulation of high levels of the envelope precursor glycoprotein (gPr80env
) in the endoplasmic reticulum (ER), which results in ER stress and apoptosis (7
). Previous studies of superinfection related to cell killing involved virus infection of cells, which results in the production of high levels of both unintegrated viral DNA and the envelope protein; thus, no conclusions could be drawn about whether both viral products are essential for cell killing or whether only one of them is sufficient. Because of our previous detection of the accumulation of high levels of gPr80env
in cells that are undergoing apoptosis by MCF13 MLV infection (19
), we undertook this study to determine whether cell killing is inducible by the envelope protein alone and whether ER stress is involved.
For exogenous expression of the MCF13 MLV envelope protein, we produced a plasmid clone consisting of the env
gene of this retrovirus. Because we previously observed that the xenotropic MLV strain NZB-9 did not induce either apoptosis or ER stress in virus-infected cells (18
), we also cloned the env
gene of this virus for comparison. The envelope glycoproteins of MCF13 and NZB-9 MLV were expressed in 1.6 × 106
mink epithelial cells by transient transfection of 25 μg of plasmid DNA with the use of Lipofectamine 2000 according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). Intracellular Env expression in transfected cells was detected by an indirect immunofluorescence assay in which the primary antibody was an MLV Env-specific monoclonal antibody (MAb), MAb 83A25 (9
). Cells expressing the envelope protein were enumerated at 48 h after transfection, because this was when maximum expression of Env was detectable by Western blot analysis, as described below. Examination of 230 to 607 transfected cells by immunofluorescence microscopy in each of seven independent experiments indicated that average percentages of 25.6% and 21.1% of the transfected cells expressed the envelope proteins for MCF13 and NZB-9, respectively. Thus, the efficiencies of transfection were comparable for both env
gene plasmids. Similar transfection efficiencies were also obtained when an expression plasmid for β-galactosidase was cotransfected as an additional control (data not shown).
To examine steady-state levels of the polyprotein precursor (gPr80env
) and cleaved surface (SU) forms of Env for each virus, we performed Western blot analysis of cellular extracts isolated from transfected cells. At 24, 48, and 72 h after transfection, we observed that the predominant form of the MCF13 envelope was the gPr80env
precursor, which was present at levels approximately 20- to 40-fold greater than that of SU (Fig. , lanes 4, 7, and 10). In contrast, we detected nearly equivalent amounts of gPr80env
and SU for NZB-9, with a precursor to SU mean ratios that ranged from 1.9 at 24 h to 0.8 at 72 h posttransfection (Fig. , lanes 5, 8, and 11). Analysis of the protein band intensities of all Western blots was performed with a Kodak EDAS 120 scanner and software. Slight differences in mobility between the MCF13 Env precursor and SU proteins and that of the corresponding NZB-9 glycoproteins were detectable, similar to what has been described for MCF247 MLV and the xenotropic 69X9 MLV (10
). The results of pulse-chase analyses of envelope proteins for both viruses indicated that processing of the MCF13 polyprotein precursor occurred more slowly and incompletely than that of the NZB-9 polyprotein (Fig. ). For these assays, we used virus-infected cells because they contained greater amounts of envelope protein for immunoprecipitation.
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 (more ...)