Transmissible gastroenteritis virus N protein causes endoplasmic reticulum stress, up-regulates interleukin-8 expression and its subcellular localization in the porcine intestinal epithelial cell

Abstract

This essay focuses on transmissible gastroenteritis virus (TGEV), which is an enteropathogenic virus related to contagious and acute diseases in suckling piglets. Previous literature suggests that the TGEV nucleocapsid protein (N) plays a significant role in viral transcriptional process, however, there is a need to examine other functions of TGEV N protein in the porcine intestinal epithelial cell (IEC) which is the target cell of TGEV. In the present study, we investigated the degradation, subcellular localisation, and function of TGEV N protein by examining its effects on cycle progression, endoplasmic reticulum (ER) stress, interleukin-8 (IL-8) expression, and cell survival. The results showed that TGEV N protein localised in the cytoplasm, inhibited IEC growth, prolonged the S-phase cell cycle by down-regulating cell cycle protein cyclin A, and was mainly degraded through the proteasome pathway. Moreover, TGEV N protein induced ER stress and activated NF-κB, which was responsible for the up-regulation of IL-8 and Bcl-2 expression. This report mainly considers the functions of TGEV N protein in IEC. To be specific, in IEC, TGEV N protein induces cell cycle prolongation at the S-phase, ER stress and up-regulates IL-8 expression. These results provide a better understanding of the functions and structural mechanisms of TGEV N protein.

Keywords: ER stress; IL-8; Location; N protein; NF-κB; TGEV.

Fig. 1

The expression products and protein degradation characteristics of TGEV N protein in IEC. Cells were transfected with N-GFP expression vector or GFP vector and treated with MG132 for 24 h. (A) The cells were subjected to Western blot analysis using anti-GFP antibodies. (B) The cells were subjected to Western blot analysis using TGEV N protein antibodies. (C) Protein degradation characteristics were observed by fluorescence microscopy. All the data shown are representative of three independent experiments.

Fig. 2

Subcellular localization of TGEV N protein in IEC. Cells were transfected with N-GFP expression vector or GFP vector. (A) Cells were stained by Hoechst33342 and ER-Tracker ™ Red. (B) Cells were stained with anti-N antibody, followed by goat anti-mouse antibody. Bar = 20 μm for all the figures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Cell cycle arrest and the expression of cyclin A induced by TGEV N protein. Cells were transfected with N-GFP expression vector or GFP ector for 48 h. (A) Flow cytometry analysis of cells by propidium iodide staining. (B) The percentage of cells in each phase of the cell cycle from flow cytometry data. (C) The level of cyclin A expression was determined by western blot. β-actin was used as an internal loading control. (D) Real-time PCR analysis of cyclin A mRNA levels were normalized to the corresponding CT value for porcine β-actin mRNA. The results are mean ± SD from three independent experiments. * p < 0.05 versus the control groupversus the control group (the cells expressing GFP and untransfected IEC cells).

Fig. 4

The effect of TGEV N protein in IEC on NF-κB activity and the expression of GRP78, IL-8, Bcl-2, IL-8. Cells expressing GFP or N-GFP for 48 h. (A) The level of GRP78 expression was determined by western blot. (B) Real-time PCR analysis of GRP78 mRNA levels. (C) The level of NF-κB expression was determined by western blot. (D) NF-κB p65 activation was determined using the ELISA assay. (E) The expression of IL-8 in N-GFP expressing IEC or untransfected cells (treated or untreated with MG132) culture supernatants were measured by ELISA. (F) IL-8 mRNA levels were analysed by Real-time PCR assay. (G) The level of Bcl-2 expression was determined by western blot. (H) Real-time PCR analysis of Bcl-2 mRNA levels. β-actin was used as an internal loading control. The results are mean ± SD and representative of three independent experiments.