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. 2023 Mar 3;24(5):4936.
doi: 10.3390/ijms24054936.

Porcine Epidemic Diarrhea Virus and Its nsp14 Suppress ER Stress Induced GRP78

Wei Zeng  1   2 , Jingping Ren  1   2 , Gan Yang  1   2 , Changsheng Jiang  1   2 , Ling Dong  1   2 , Qi Sun  1   2 , Yaofang Hu  1   2 , Wentao Li  1   2 , Qigai He  1   2
Affiliations

Porcine Epidemic Diarrhea Virus and Its nsp14 Suppress ER Stress Induced GRP78

Wei Zeng et al. Int J Mol Sci. .

Abstract

Porcine epidemic diarrhea virus (PEDV), a member of the α-coronavirus genus, can cause vomiting, diarrhea, and dehydration in piglets. Neonatal piglets infected with PEDV have a mortality rate as high as 100%. PEDV has caused substantial economic losses to the pork industry. Endoplasmic reticulum (ER) stress, which can alleviate the accumulation of unfolded or misfolded proteins in ER, involves in coronavirus infection. Previous studies have indicated that ER stress could inhibit the replication of human coronaviruses, and some human coronaviruses in turn could suppress ER stress-related factors. In this study, we demonstrated that PEDV could interact with ER stress. We determined that ER stress could potently inhibit the replication of GⅠ, GⅡ-a, and GⅡ-b PEDV strains. Moreover, we found that these PEDV strains can dampen the expression of the 78 kDa glucose-regulated protein (GRP78), an ER stress marker, while GRP78 overexpression showed antiviral activity against PEDV. Among different PEDV proteins, PEDV non-structural protein 14 (nsp14) was revealed to play an essential role in the inhibition of GRP78 by PEDV, and its guanine-N7-methyltransferase domain is necessary for this role. Further studies show that both PEDV and its nsp14 negatively regulated host translation, which could account for their inhibitory effects against GRP78. In addition, we found that PEDV nsp14 could inhibit the activity of GRP78 promotor, helping suppress GRP78 transcription. Our results reveal that PEDV possesses the potential to antagonize ER stress, and suggest that ER stress and PEDV nsp14 could be the targets for developing anti-PEDV drugs.

Keywords: ER stress; GRP78; PEDV; nsp14.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Figures

Figure 1
Figure 1
ER stress inhibits PEDV propagation. (a,b) Cell viability was measured by CCK8 assay at 48 h after treatment with TM or Tg. The cell viability of the mock group was used to normalize the results of other test groups. (c–h) Cells were pretreated with TM or Tg for 8 h before PEDV challenge (0.1 MOI). Samples were collected at 36 hpi or indicated time points. DMSO served as the treatment control. (c) The titer of PEDV strain DR13 determined in Tg pretreated Vero cells; (d) The titer of PEDV strain DR13 determined in TM pretreated Vero cells; (e) Viral titer determined in PEDV strain YN15 infected Vero cells; (f) The amount of PEDV N protein in PEDV strain JS infected Vero cells; (g) The amount of PEDV N protein in PEDV strain YN15 infected LLC-PK1 cells; (h) The amount of PEDV N protein in PEDV strain JS infected LLC-PK1 cells. β-actin was set as the loading control. (i,j) Vero cells were transfected with pCAGGS GRP78-flag or the control vector and exposed to 0.1 MOI PEDV 36 h after transfection. Samples were harvested at 36 hpi. The amount of PEDV was determined using western blot and RT-qPCR, respectively. “+” means that the corresponding material in the row has been added, and “−” means that the material is not added. Data represent the mean ± standard deviation (SD) of three independent experiments, and error bars represent the standard deviation. Asterisks indicate significant differences: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.
Figure 2
Figure 2
PEDV suppresses GRP78 expression. (a) Vero cells were challenged with 0.1 MOI PEDV. The amount of GRP78 protein and PEDV N protein were measured using Western blot; (b) Vero cells were first challenged with PEDV (0.1 MOI). TM (0.4 µM) or Tg (0.8 µM) was added to the culture medium at 24 hpi. Samples were harvested at 32 hpi and subjected to western blot analysis t; (c–g) Cells were first challenged with PEDV (0.01 MOI or 0.1 MOI). TM (0.4 µM) or Tg (0.8 µM) was added to the culture medium at 24 hpi. Samples were harvested at 32 hpi and subjected to western blot analysis; (h) Vero cells were transfected with pCAGGS GRP78-flag or the control vector and exposed to 0.1 MOI PEDV 36 h after transfection. Samples were harvested at 36 hpi and subjected to western blot analysis. β-actin was set as the loading control. “+” means that the corresponding material in the row has been added, and “−” means that the material is not added. Data represent the mean ± standard deviation (SD) of three independent experiments, and error bars represent the standard deviation.
Figure 3
Figure 3
PEDV nsp14 inhibits GRP78. (a) The viability of HEK293t cells was measured by CCK8 assay at 48 h after treatment with TM or Tg. The cell viability of the mock group was used to normalize the results of other test groups. (b) The effect of different PEDV proteins on GRP78 at the protein level. HEK293t cells were transfected with plasmids and treated with 0.8 µM TM 36 h after transfection. Samples were harvested 44 h after transfection and subjected to western blot analysis; (c) The effect of different PEDV proteins on GRP78 at the transcription level. Samples were harvested 36 h after transfection and subjected to RT-qPCR analysis; (d,e) HEK293t cells were transfected with pCAGGS nsp14-Flag or the control vector and treated with TM (0.8 µM) or Tg (0.8 µM) 36 h after transfection. Samples were harvested 44 h after transfection and subjected to western blot analysis; (f) Vero cells were transfected with pCAGGS nsp14-Flag or the control vector and treated with TM (0.4 µM) or Tg (0.8 µM) 36 h after transfection. Samples were harvested 44 h after transfection and subjected to western blot analysis. β-actin was set as the loading control. Data represent the mean ± standard deviation (SD) of three independent experiments, and error bars represent the standard deviation. “+“ means that the corresponding material in the row has been added, and “−” means that the material is not added.
Figure 4
Figure 4
PEDV nsp14 N7-MTase domain is crucial for inhibiting GRP78. (a,b) Plasmids transfected HEK293t cells were treated with TM (0.4 µM) or Tg (0.8 µM), respectively. Western blot was performed to determine the expression level of proteins. β-actin was set as the loading control. “+” means that the corresponding material in the row has been added, and “−” means that the material is not added.
Figure 5
Figure 5
PEDV and its nsp14 inhibit cellular translation. (a) Vero cells were challenged with 0.1 MOI PEDV and subjected to puromycin incorporation assay at 36 hpi. Lanes 3 and 6 are repetitions of lanes 2 and 5, respectively; (b) HEK293t cells were transfected with pCAGGS nsp14-Flag or the control vector. Cells were subjected to puromycin incorporation assay 36 h after transfection. β-actin was set as the loading control. “+” means that the corresponding material in the row has been added, and “−” means that the material is not added.
Figure 6
Figure 6
PEDV nsp14 subcellular localization. (a,b) Vero cells and HEK293t cells were transfected with pCAGGS nsp14-Flag or the control vector. Samples were harvested 36 h after transfection and subjected to IFA analysis. Images were generated using a confocal laser scanning microscope. Scale bar is 30 µm.
Figure 7
Figure 7
PEDV nsp14 inhibits the activity of GRP78 promoter. (a) Putative sequences for the GRP78 promoter were predicted using Promoter 2.0 Prediction Server; (b) Predicted sequences possess positive promoter activity. HEK293t cells were transfected with plasmids and subjected to luciferase reporter assay 36 h after transfection; (c,d) The pCAGGS nsp14-Flag or the control vector was co-transfected with the human-GRP78-promoter-mRFP, the porcine-GRP78-promoter-mRFP, and the mRFP control vector, respectively. Samples were harvested 36 h after transfection. Flow cytometry was performed to analyze mRFP signal positive cells ratio and the mean fluorescence intensity of mRFP positive cells. Data represent the mean ± standard deviation (SD) of three independent experiments, and error bars represent the standard deviation. Asterisks indicate significant differences: *** p < 0.001; **** p < 0.0001; ns, not significant.
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