Metalloprotease ADAM17 regulates porcine epidemic diarrhea virus infection by modifying aminopeptidase N

Abstract

Porcine epidemic diarrhea virus (PEDV) is a causative agent of porcine epidemic diarrhea (PED). PED, characterized by acute diarrhea, vomiting, dehydration, has caused serious economic losses in pig industry worldwide. Here, we demonstrate that activation of a disintergrin and metalloprotease 17 (ADAM17) induced the decrease of PEDV infection in HEK293 and IPEC-J2 cells and the downregulation of cell surface aminopeptidase N (APN) expression, an important entry factor for PEDV infection. Furthermore, overexpression of ADAM17 suppressed PEDV infection in HEK293 and IPEC-J2 cells, whereas ablation of ADAM17 expression using ADAM17 specific siRNA resulted in a corresponding increase of PEDV infection and an upregulation of cell surface APN expression. Taken together, these data demonstrate that modulation of APN expression by metalloprotease ADAM17 regulates PEDV infection. Hence, the reduction in APN expression represents another component of the anti-PEDV infection response initiated by ADAM17.

Keywords: ADAM17; Aminopeptidase N; PEDV.

Fig. 1

Effect of the metalloprotease inhibitor BB94 and activator PMA on PEDV infection. (A) HEK293 cells were treated with metalloprotease inhibitor BB94 or DMSO (carrier control) at the concentration of 2 μM for 24 h followed by PEDV infection at MOI of 0.1. At 24 h after infection, cells were lysed and subjected to western blot with mAb 2G3 against PEDV N protein. Meanwhile, cell samples were collected and titered by TCID₅₀. (B) Role of metalloprotease activator PMA on PEDV infection in HEK293 cells. HEK293 cells were treated with metalloprotease activator PMA or DMSO at the concentration of 2 μM for 30 min followed by infection with PEDV at MOI of 0.1. At 24 h post infection, cells were collected and analyzed as indicated above. (C) Role of ADAM17 on PEDV infection in IPEC-J2 cells. IPEC-J2 cells were treated with BB94 or PMA as indicated for HEK293 and subjected to PEDV infection at MOI of 1.0. Cells were collected and subjected to progeny titration by TCID₅₀. Data represent the means of three independent experiments (± SD) of virus titers. *, p< 0.05. The p value is calculated using Student's t-test. (D) Expression level of ADAM17 in PMA, BB94, DMSO, and mock -treated cells. HEK293 cells were treated with PMA, BB94, DMSO, and mock, and cell lysates were subjected to western-blot with antibodies as indicated.

Fig. 2

ADAM17 suppresses PEDV infection in HEK293 and IPEC-J2 cells. (A&C) Overexpression of ADAM17 reduces PEDV infection. Cells were transfected with pCAGGS/ADAM17 (ADAM17) or pCAGGS vector control (Mock) for 24 h followed by infection with PEDV at MOI of 0.1 (HEK293) or 1.0 (IPEC-J2). At 24 h after infection, cells were lysed and subjected to western blot with mAb 2G3 against PEDV N protein and mAb anti-Flag to detect ADAM17. At the same time, virus samples were collected and titered. (B&D) Knockdown of endogenous ADAM17 facilitates PEDV infection. Cells were transfected with ADAM17-specific siRNA (siADAM17) or scramble control siRNA (siNC) for 24 h followed by infection with PEDV at MOI of 0.1 (HEK293) or 1.0 (IPEC-J2). At 24 h after infection, cells were collected and subjected to western blot and virus titration as indicated. Data represent the means of three independent experiments (± SD) of virus titers. *, p< 0.05. The p value is calculated using Student's t-test.

Fig. 3

ADAM17-mediated APN modulation regulates PEDV infection in HEK293 cells. (A) HEK293 cells were treated with PMA for 30 min, and cells were then transfected with vector or APN for 24 h followed by PEDV infection. Virus titers were determined by TCID₅₀. HEK293 cells were treated with inhibitor BB94 (B) and activator PMA (C) for 24 h and 30 min, respectively. All cells were then stained for surface expression of APN with mouse anti-APN antibody and detected by flow cytometry. Moreover, HEK293 cells were transfected with pCAGGS/ADAM17 (ADAM17) and pCAGGS vector control (D), or ADAM17-specific siRNA duplexes (siADAM17, 100 nM) and negative control siRNA (siNC) (E) as indicated. Levels of APN expression were determined by flow cytometry. Appropriate isotype negative control antibodies were selected as control. The data are the representatives from three independent measurements.