Epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry

Abstract

Transmissible gastroenteritis virus (TGEV) causes severe diarrhea and high mortality in newborn piglets. It is well established that porcine intestinal epithelium is the target of the TGEV infection, however the mechanism that TGEV invades the host epithelium remains largely unknown. Aminopeptidase N (APN) is a known receptor of TGEV. This study discovered that the extracellular receptor binding domain 1 pertaining to epidermal growth receptor (EGFR) interact with TGEV spike protein. APN and EGFR synergistically promote TGEV invasion. TGEV promotes APN and EGFR clustering early in infection. Furthermore APN and EGFR synergistically stimulate PI3K/AKT as well as MEK/ERK1/2 endocytosis signaling pathways. TGEV entry is via clathrin and caveolin mediated endocytosis in IPEC-J2 cells. TGEV binds with EGFR, and subsequently promotes EGFR internalization by a clathrin-mediated endocytosis pathway. These results show that EGFR is a co-factor of TGEV, and that it plays a synergistic role with APN early in TGEV infection.

Keywords: Aminopeptidase N; Caveolin; Clathrin; Epidermal growth receptor; IPEC-J2 cells; TGEV.

Fig. 1

Interaction between TGEV S1 protein and EGFR extracellular receptor binding domain 1. (A) Structure of EGFR. (B) His-tagged EGFR extracellular receptor binding domain 1 or 2 expressed in E.coli BL21 and purified in Ni-NTA columns, the purified products were separated using SDS-PAGE and stained with Coomassie brilliant blue. (C) The purified EGFR extracellular receptor-binding domain 1 or 2 were verified by Western-blot. (D) TGEV (MOI = 2) was incubated in DMEM containing His-32a, His-EGFR Receptor 1 or His-EGFR Receptor 2 at 37 °C for 2 h, then incubated with IPEC-J2 cells and cultured for 1 h. The invasion of TGEV was detected by RT-PCR. (E) TGEV (MOI = 2) was incubated in DMEM containing His-32a, His-EGFR Receptor 1 and His-EGFR Receptor 2 at 37 °C for 2 h, then incubated with IPEC-J2 cells, and cultured for 1 h, the viral titers of intracellular TGEV were analyzed by tissue culture infectivity dose 50 TCID₅₀. (F) IPEC-J2 cells were pretreated with His-EGFR Receptor 1 at different concentrations at 37 °C for 2 h, then incubated with IPEC-J2 cells, and cultured for 1 h. The invasion of TGEV was detected by RT-PCR. (G) Intracellular TGEV were analyzed by viral plaque morphology in ST cells. (H) The lysates of TGEV-infected IPEC-J2 cells were immunoprecipitated with rabbit anti-EGFR or normal Rabbit IgG. Immunoblotting was then performed to determine the presence of EGFR and TGEV in the EGFR immunoprecipitate. (I and J) 293T cells were co-transfected with a HA-tagged TGEV S1 expression plasmid together with GFP-tagged EGFR Receptor 1 or GFP-tagged EGFR Receptor 2 expression plasmid, cell lysates were immunoprecipitated with an anti-HA antibody or an anti-GFP antibody, the resulting precipitates were examined by immunoblotting using an anti-HA or an anti-GFP antibody to examine the interaction between HA-TGEV S1 and GFP-tagged EGFR. (** p < 0.01).

Fig. 2

TGEV infection causes the co-localization of APN and EGFR. IPEC-J2 cells were infected with TGEV (MOI = 2) and cultured for 30 min, then stained for fluorescence microscopy using mouse anti-APN pAb, followed by DyLight 488-conjugated goat anti-mouse IgG and rabbit anti-p-EGFR mAb, followed by DyLight 594-conjugated goat anti-rabbit IgG. Mock-infected cells served as controls. The data shown are from two independent experiments.

Fig. 3

APN and EGFR synergistically promote TGEV invasion. (A and B) APN and EGFR interference verification, shAPN1 and shEGFR3 were later used in the subsequent experiments. (C) IPEC-J2 cells were transfected with interference vector pLVX-shRNA-APN, pLVX-shRNA-APNCtrl, pLVX-shRNA-EGFR, pLVX-shRNA-EGFRCtrl, or pLVX-shRNA-APN + pLVX-shRNA-EGFR through lentiviral supernatant. Normal cells served as controls. Cells were infected with TGEV at an MOI of 2, and cultured for 1 h. The invasion of TGEV was detected by RT-PCR. (D and E) IPEC-J2 cells were infected with Dylight 488-TGEV, and cultured for 1 h The invasion of TGEV was detected by Flow cytometry. (F) The viral titers of intracellular TGEV were analyzed by TCID₅₀. (G) Intracellular TGEV was analyzed by viral plaque morphology in ST cells. The data shown are the mean results ± SD from three independent experiments. (* 0.01 < p < 0.05, ** p < 0.01).

Fig. 4

APN and EGFR synergistically activate PI3K/AKT and MEK/ERK1/2 signaling pathways. (A) IPEC-J2 cells were transfected with overexpression vector pLVX-DsRed, pLVX-APN-DsRed, pLVX-EGFR-DsRed, or pLVX-APN-DsRed + pLVX-EGFR-DsRed through lentiviral supernatant. Cells were infected with TGEV at an MOI of 2 and cultured for 30 min. Normal cells and TGEV infected-normal cells served as controls. The activation of downstream signaling pathways analyzed by Western-blot with anti-p-EGFR, anti-EGFR, anti-p-AKT, anti-AKT, anti-p-ERK1/2, anti-ERK1/2, and anti-GAPDH antibodies. (B) IPEC-J2 cells were transfected with interference vector pLVX-shRNA-APN, pLVX-shRNA-APNCtrl, pLVX-shRNA-EGFR, pLVX-shRNA-EGFRCtrl, or pLVX-shRNA-APN + pLVX-shRNA-EGFR through lentiviral supernatant. Cells were infected with TGEV at an MOI of 2, and cultured for 30 min Normal cells and TGEV infected-normal cells served as controls. The activation of downstream signaling pathways analyzed by Western-blot with anti-p-EGFR, anti-EGFR, anti-p-AKT, anti-AKT, anti-p-ERK1/2, anti-ERK1/2, and anti-GAPDH antibodies. (C–H) The ratio of p-EGFR, EGFR, p-AKT, AKT, p-ERK1/2 or ERK1/2 to GAPDH was normalized to control conditions. Data shown are the mean results ± SD from three independent experiments. (* 0.01 < p < 0.05, ** p < 0.01).

Fig. 5

Clathrin and Caveolin mediate the endocytosis of TGEV. (A and B) Clathrin and Caveolin interference verification, shClai3 and shCav2 were later used in the subsequent experiments. (C and D) IPEC-J2 cells were transfected with interference vector pLVX-shRNA-Clathrin, pLVX-shRNA-ClathrinCtrl, pLVX-shRNA-Caveolin, or pLVX-shRNA-CaveolinCtrl through lentiviral supernatant. Normal cells served as controls. Cells were infected with TGEV at an MOI of 2 and cultured for 1 h. The invasion of TGEV was detected by RT-PCR. (E–G) IPEC-J2 cells were transfected with interference vector pLVX-shRNA-Clathrin, pLVX-shRNA-ClathrinCtrl, pLVX-shRNA-Caveolin, or pLVX-shRNA-CaveolinCtrl through lentiviral supernatant. Cells were infected with TGEV at an MOI of 2, and cultured for 1 h. The invasion of TGEV was detected by Flow cytometry (E and F). The viral titers of intracellular TGEV were analyzed by TCID₅₀ (F). (H and I) The co-localization of transferrin and cholera toxin with TGEV, (scale bar = 20 µm), the immunofluorescence experiment was repeated two times, every time there are three groups of parallel samples. The data shown are the mean results ± SD from three independent experiments. (* 0.01 < p < 0.05, ** p < 0.01).

Fig. 6

TGEV infection induced EGFR internalization. (A) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. Then stained for fluorescence microscope using rabbit anti-EGFR pAb followed by DyLight 594-conjugated goat anti-rabbit IgG, Mock-infected cells served as controls. EGFR distribution was observed by confocal microscope. (B) Three-dimensional rendering of representative images obtained using Imaris 7.2 software. (C) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. The protein of the cell membrane was extracted. Cell membrane EGFR was analyzed by Westernblot using rabbit anti-EGFR pAb. (D) The ratio of EGFR to the mean of E-cadherin and GAPDH was normalized to control conditions. The data shown are the mean results ± SD, from three independent experiments. (scale bar = 20 µm).

Fig. 7

EGFR internalization through clathrin endocytosis pathway. (A) IPEC-J2 cells were transfected with interference vector pLVX-shRNA-Clathrin3 or pLVX-shRNA-Caveolin2 through lentiviral supernatant. Normal cells served as controls. Cells were infected with TGEV at an MOI of 2. The protein of the cell membrane was extracted. Cell membrane EGFR was analyzed by Westernblot using rabbit anti-EGFR pAb. (B) The ratio of EGFR to the mean of E-cadherin and GAPDH was normalized to control conditions. The data shown are the mean results ± SD from three independent experiments.

Fig. 8

APN and EGFR synergistically promote TGEV invasion. EGFR is a co-factor for TGEV invasion. TGEV S1 protein interacts with EGFR extracellular receptor binding domain 1. TGEV infection induces EGFR internalization and causes APN and EGFR clustering. APN and EGFR synergistically promote TGEV invasion. APN and EGFR synergistically activate PI3K/AKT and MEK/ERK1/2 signaling pathways. Clathrin and caveolin mediate the endocytosis of TGEV and EGFR internalization through clathrin endocytosis pathway.

Fig. S1

APN and EGFR overexpression verification.