Induction of endoplasmic reticulum stress in thymic lymphocytes by the envelope precursor polyprotein of a murine leukemia virus during the preleukemic period

Abstract

Infection of thymic lymphocytes by a mink cell focus-forming murine leukemia virus induces apoptosis during the preleukemic period of lymphomagenesis. In this study, we observed that during this period, the viral envelope precursor polyprotein accumulated to high levels in thymic lymphocytes from mice inoculated with virus. Envelope accumulation occurred with the same kinetics as the induction of endoplasmic reticulum (ER) stress, which resulted in the upregulation of the 78-kDa glucose-regulated protein (GRP78). In thymic lymphomas, GRP78 levels were higher than those in virus-infected preleukemic cells, and GRP58 was upregulated. These results suggest that Env precursor accumulation induces ER stress, which participates in thymic lymphocyte apoptosis. The subsequent upregulation of ER chaperone proteins GRP78 and GRP58 may contribute to rescuing cells from virus-induced apoptosis.

FIG. 1.

Time course analysis of ER stress-associated chaperones in thymic lymphocytes after inoculation of MCF13 MLV into neonatal mice. Western blot analysis was carried out with protein extracts from thymic lymphocytes isolated from mice at different times after virus inoculation. C1 to C5, age-matched control mice inoculated with medium; M1 to M9, mice inoculated with virus at 2 to 4 days of age. ER chaperone proteins GRP78 (A), GRP58 (B), and GRP94 (C) were detected with specific antibodies (anti-GRP78, Santa Cruz Biotechnology, Inc.; anti-GRP58 and anti-GRP94, Stressgen Biotechnologies). α-Tubulin was detected as a control for loading.

FIG. 2.

Viral glycoprotein detection in thymic lymphocytes. Western blot analysis of protein extracts from preleukemic thymic lymphocytes at different times after virus inoculation (A) or from thymic tumors and a tumor-derived cell line (B). C1 to C7, age-matched control mice that were inoculated with medium. M1 to M9, mice inoculated with virus at 2 to 4 days of age. Mink, protein extracts from productively infected mink epithelial cells used as markers for the mobility of the Env precursor polyprotein gPr80^env and SU gp70. The ratio of these proteins in productively infected cells is different from that in acutely infected cells, in which gPr80^env predominates (14). T1 to T7, thymic lymphomas induced by inoculation of MCF13 MLV. T8 to T11, spontaneous tumors arising in uninoculated mice. 92316, T-cell line derived from a thymic lymphoma induced by MCF247 MLV. MCF MLV Env was detected with the MAb 83A25 (5). α-Tubulin was detected as a control for loading.

FIG. 3.

Detection of GRPs in thymic lymphoma cells. Western blotting was performed as described for Fig. 1. GRP78 (A), GRP58 (B), and GRP94 (C) were detected with specific antibodies. C1 and C2, age-matched control mice inoculated with medium; T1 to T7, thymic lymphomas induced by inoculation of MCF13 MLV into neonatal mice; T8 to T11, spontaneous tumors arising in uninoculated mice; 92316, T-cell line derived from a thymic lymphoma induced by MCF247 MLV. α-Tubulin was detected as a loading control.