Up-regulation of a cellular protein at the translational level by a retrovirus

Abstract

Mink cell focus-forming (MCF) murine leukemia viruses (MLVs) are the etiologic agent of thymic lymphoma in mice. We have observed previously that superinfection by MCF13 MLV of certain cell types, such as preleukemic thymic lymphocytes and cultured mink epithelial cells, results in the accumulation of the viral envelope precursor polyprotein, leading to the induction of endoplasmic reticulum (ER) stress. In this study, we demonstrate that the induction of ER stress by MCF13 MLV infection results in an increase in the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2. In cells in which this occurs, we have detected an up-regulation of the cellular inhibitor of apoptosis protein 1 (c-IAP1). The results of real-time RT-PCR quantification of message levels and protein turnover assays indicate that up-regulation of c-IAP1 occurs at the translational level. Elevation of c-IAP1 levels at a posttranscriptional step was detectable in MCF13 MLV-induced thymic lymphomas and chronically infected mink epithelial cells. The ability of a simple retrovirus to regulate cellular gene expression at the translational level may be an important mechanism that contributes to pathogenesis.

Fig. 1.

Up-regulation of eIF2α phosphorylation in cells that undergo ER stress by MLV infection. (A) Immunoblot of eIF2α-P in mink epithelial cells either mock-infected as control (C) or infected with either MCF13 MLV (M) or NZB-9 MLV (N) that was detectable with an anti-eIF2α-P that specifically recognizes phosphorylation at Ser-51 (ab4837; Abcam). (B) Total eIF2α in the same cells as described in A that was detectable with anti-total eIF2α (ab5369; Abcam). (C) eIF2α-P in M. dunni fibroblasts either mock-infected (C) or infected with MCF13 MLV (M). (D) Total eIF2α in the same cells as in C. All virus infections were performed at a MOI of 1. Cellular protein was analyzed at different days p.i. α-Tubulin or β-actin was detected as a loading control.

Fig. 2.

ER stress-induced proteins in virus-infected cells. (A) Immunoblots for GRP78 in mink epithelial cells either mock-infected as control (C) or infected with either MCF13 (M) or NZB-9 (N) MLV. (B) CHOP for the same protein samples shown in A. (C) GRP78 in M. dunni fibroblasts that were infected with MCF13 MLV (M) or mock-infected (C). (D) CHOP for the same protein samples shown in C. Minor bands seen in B are a result of nonspecific antibody binding. Virus infections were performed at a MOI of 1. Cellular protein was analyzed at different days p.i. α-Tubulin or β-actin was detected as a control for loading.

Fig. 3.

c-IAP1 protein up-regulation in MCF13 MLV-infected mink cells. Immunoblots of c-IAP1 in mink epithelial cells either mock-infected as control (C) or infected with MCF13 MLV (M) or NZB-9 MLV (N). (A) MCF13 MLV infection at a MOI of 20. Mink cells either untreated (Un, lane 13) or treated with tunicamycin (Tm, lane 14) or thapsigargin (Tg, lane 15). (B) MCF13 or NZB-9 MLV infection at a MOI of 1. (C) c-IAP1 in M. dunni fibroblasts either mock-infected (C) or infected with MCF13 MLV (M) at a MOI of 1. Histograms below each immunoblot show the ratio of intensity of the c-IAP1 protein band for virus-infected cells vs. that of the respective control cells as fold-increase. Bars represent cells infected with either MCF13 MLV (unshaded) or NZB-9 MLV (shaded). Cellular protein was analyzed at different days p.i. α-Tubulin or β-actin was detected as a loading control.

Fig. 4.

c-IAP1 up-regulation occurs at the translational level. (A) Real-time qRT-PCR analysis of c-IAP1 mRNA in mink epithelial cells either mock-infected (open bar) or MCF13 MLV-infected (shaded bar) at days 5 and 11 p.i. Mean values and standard errors of the mean were calculated from assays of duplicate or triplicate samples from each of two independent experiments (n = 4–6). Results are expressed relative to the level of c-IAP1 mRNA in control cells. (B and C) Immunoprecipitated c-IAP1 after a 30-min pulse with [³⁵S]Met-Cys and chase for various times from a representative experiment for mock-infected (B) or MCF13 MLV-infected (C) cells. Serum lane is immunoprecipitation control with normal rabbit serum. (D) ³⁵S-labeled protein extracts prepared from mock-infected (C) or MCF13 MLV-infected (M) mink cells were subjected to SDS/PAGE. α-Tubulin was detected by ECL as a loading control. Migration of marker proteins with known molecular masses (kDa) is shown at left.

Fig. 5.

Elevation of c-IAP1 in mink cells chronically infected with MCF13 MLV and in virus-induced thymic lymphomas at the posttranscriptional level. (A and B) Detection of c-IAP1 protein by immunoblotting for mink cells either uninfected as control (C) or chronically infected with MCF13 MLV (M) and mink cells either untreated (Un) or treated with tunicamycin (Tm) or thapsigargin (Tg) at 1 μg per ml for 18 h (A) and control thymus (C), thymic tumors (T1-T4), or NIH 3T3 fibroblasts that were either untreated (Un) or treated with tunicamycin (Tm) (B). α-Tubulin was detected as a loading control. (C and D) Real-time qRT-PCR analysis of c-IAP1 mRNA in uninfected (C) and chronically infected (M) mink cells shown in A (C) and control thymus and thymic tumors T2-T4 shown in B (D). Mean values calculated from triplicate assays and their ranges are indicated for each cell type. P values that were determined by Student's t test are 0.6 for the comparison between uninfected and chronically infected cells shown in C and 0.09–0.8 for comparisons between the control thymus and thymic tumors shown in D.