PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway

Abstract

With the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (PEDV) has led to significant economic loss in the global swine industry. Many studies have described how coronaviruses enter cells, but information on PEDV invasion strategies remains insufficient. Given that the differences in gene sequences and pathogenicity between classical and mutant strains of PEDV may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the PEDV GI and GII subtype strains in Vero cells and IPEC-J2 cells. We first characterized the kinetics of PEDV entry into cells and found that the highest invasion rate of PEDV was approximately 33% in the IPEC-J2 cells and approximately 100% in the Vero cells. To clarify the specific endocytic pathways, systematic research methods were used and showed that PEDV enters cells via the clathrin- and caveolae-mediated endocytosis pathways, in which dynamin II, clathrin heavy chain, Eps15, cholesterol, and caveolin-1 were indispensably involved. In addition, lipid raft extraction assay showed that PEDV can also enter cells through lipid raft-mediated endocytosis. To investigate the trafficking of internalized PEDV, we found that PEDV entry into cells relied on low pH and internalized virions reached lysosomes through the early endosome-late endosome-lysosome pathway. The results concretely revealed the entry mechanisms of PEDV and provided an insightful theoretical basis for the further understanding of PEDV pathogenesis and guidance for new targets of antiviral drugs.

Figure 1

Trypsin-dependency and kinetics of PEDV entry into cells. A Vero cells were seeded in 6-well plates until confluence. Cells were washed with PBS and infected with PEDV strains (MOI = 0.5) without trypsin or in the presence of trypsin (10 μg/mL) or trypsin and 25 μg/mL SBTI. Cells were collected for qRT-PCR at 12 hpi. B, C Vero cells (B) and IPEC-J2 cells (C) were incubated with PEDV GDS01 and GDS09 strains, respectively, at 4 °C for 1 h and shifted to 37 °C immediately to initiate internalization. At 0, 15, 30, 45, 60, 75, 90, 105, and 120 min after incubation, the cells were treated with proteinase K (1 mg/mL) at 4 °C for 30 min to inactivate the non-internalized virions. The control cells were washed with PBS. The invasion rates were calculated by qRT-PCR analysis. ****P < 0.001.

Figure 2

Dynamin II involved in PEDV entry. A Cells were pre-treated with 30 μM and 50 μM dynasore at 37 °C for 1 h, respectively, and incubated with GDS01 or GDS09 strain for 1 h. DMSO was used as a negative control. The cells were collected at 6 hpi for qRT-PCR assay to test the invasion efficiency of PEDV. B Vero and IPEC-J2 cells were transfected with GFP-Dyn-WT and GFP-Dyn-M, respectively, and infected with PEDV strains at 24 h after transfection. The cells were fixed at 12 hpi and stained for confocal analysis. C, D Vero and IPEC-J2 cells were transfected with siDyn twice and infected with PEDV strains at 24 h after the second transfection. The invasion rates of PEDV into the cells were detected at 6 hpi and 9 hpi for qRT-PCR and Western blotting analysis, respectively. Ctrl means control. Scale bars indicate 25 μm. **0.05 < P < 0.01; ***0.01 < P < 0.001; ****P < 0.001.

Figure 3

PEDV entry relies on the CME pathway. A Vero cells and IPEC-J2 cells were incubated with mixture of Alexa-594 labeled Trf (red) and PEDV (green) at 4 °C for 1 h, and then shifted to 37 °C for 30 min. The cells were fixed and stained for PEDV using monoclonal antibody against PEDV S protein. The cellular localizations of Trf and PEDV were observed with a confocal fluorescence microscope. Light exposure was avoided throughout this process. B, C. The Vero cells were pre-treated with 10 μM and 30 μM of CPZ, and the IPEC-J2 cells were pre-treated with 30 μM and 50 μM of CPZ, respectively, at 37 °C for 1 h and incubated with GDS01 or GDS09 strains for 1 h. Double-distilled water was used as a negative control. The cells were collected at 6 hpi and 9 hpi for qRT-PCR and Western blotting assay, respectively, to test the invasion efficiency of PEDV. D The Vero cells (left) and IPEC-J2 cells (right) were transfected with GFP-EPS15-WT and GFP-EPS15-M, respectively, and infected with PEDV strains at 24 h after transfection. The cells were fixed at 12 hpi and stained for confocal analysis. E–H The Vero cells and IPEC-J2 cells were transfected with siCHC and siEPS 15 and infected with PEDV strains at 24 h after the second transfection. The cells were collected at 6 hpi and 9 hpi for qRT-PCR and Western blotting analysis, respectively. I The cells were pre-cooled at 4 °C for 15 min, incubated with PEDV strains at 4 °C for 1 h, shifted to 37 °C for 5 min to initiate internalization, and washed for three times to remove un-internalized viral particles. The cells were fixed and stained with anti-PEDV-S (red) and anti-CHC (green) primary antibodies. Ctrl means control. Scale bars indicate 50 μm in A, 25 μm in D, and 5 μm in I. **0.05 < P < 0.01; ***0.01 < P < 0.001; ****P < 0.001.

Figure 4

PEDV entry relies on cholesterol. A, B Vero cells and IPEC-J2 cells were pre-treated with 1.5 mM and 3 mM and 1 mM and 1.5 mM MβCD, respectively, at 37 °C for 1 h and incubated with GDS01 or GDS09 strains for 1 h. Double-distilled water was used as a negative control. The cells were collected at 6 hpi and 9 hpi for qRT-PCR and Western blotting assay, respectively, to test the invasion efficiency of PEDV. C, D The Vero cells and IPEC-J2 cells were pre-treated with 10 μM and 30 μM and 30 μM and 50 μM of nystatin, respectively, at 37 °C for 1 h and incubated with GDS01 or GDS09 strains for 1 h. DMSO was used as a negative control. The cells were collected at 6 hpi and 9 hpi for qRT-PCR and Western blotting assay, respectively, to test the invasion efficiency of PEDV. E The Vero cells and IPEC-J2 cells were pre-treated with different concentrations of MβCD at 37 °C for 1 h, supplemented with 400 μg/mL of soluble cholesterol at 37 °C for 1 h, and infected with PEDV strains for 1 h. The cells were collected at 6 hpi for qRT-PCR assay to test the invasion efficiency of PEDV. *P < 0.05; **0.05 < P < 0.01; ***0.01 < P < 0.001; ****P < 0.001.

Figure 5

PEDV enters cells through the caveolae-mediated pathway. A Vero cells and IPEC-J2 cells were incubated with a mixture of Alexa-555 labeled CTB (red) and PEDV (green) at 4 °C for 1 h, and then shifted to 37 °C for 30 min. The cells were fixed and stained for PEDV using monoclonal antibody against S protein. The cellular localizations of CTB and PEDV were observed with a confocal fluorescence microscope. Light exposure was avoided throughout this process. B Vero cells (up) and IPEC-J2 cells (down) were transfected with wild-type caveolin-1 (GFP-Cav-WT) and domain negative mutant of caveolin-1 (GFP-Cav-M), respectively, and infected them with PEDV strains at 24 h after transfection. The cells were fixed at 12 hpi and stained for confocal analysis. C, D The Vero cells and IPEC-J2 cells were transfected with siCav twice and infected with PEDV strains at 24 h after the second transfection. The cells were collected at 6 hpi and 9 hpi for qRT-PCR and Western blotting analysis, respectively. Ctrl means control. E Cells were pre-cooled at 4 °C for 15 min, incubated with PEDV strains at 4 °C for 1 h, shifted to 37 °C to initiate internalization for 10 min, and washed for three times to remove viral particles that were not internalized. The cells were fixed and stained with anti-PEDV-S (red) and anti-caveolin-1 (green) primary antibodies. Scale bars indicate 50 μm in A, 25 μm in B, and 5 μm in E. *P < 0.05; **0.05 < P < 0.01; ***0.01 < P < 0.001; ****P < 0.001.

Figure 6

PEDV utilizes lipid rafts to enter cells. Vero cells (A) and IPEC-J2 cells (B) were incubated or not with PEDV at 37 °C for 1 h and then lysed in TNE buffer containing 1% Triton X-100 and 1% PMSF on ice for 30 min. After mixing with isometric 80% sucrose, the homogenized cell lysates were subjected to ultracentrifugation after being overlaid with 30% and 5% sucrose. After centrifugation, a total of 12 fractions were collected from the top to the bottom of the tubes. The localizations of the lipid raft-associated protein caveolin-1 and PEDV N protein were analyzed by Western blotting after being concentrated with 6% PEG6000.

Figure 7

PEDV entry requires low pH. A, B Vero cells and IPEC-J2 cells were pre-treated with 30 mM and 50 mM NH4Cl at 37 °C for 1 h and then incubated with GDS01 or GDS09 strains for 1 h. Double-distilled water was used as a negative control. The cells were collected 6 hpi and 9 hpi for qRT-PCR and Western blotting assay, respectively, to test the invasion efficiency of PEDV. C, D The Vero cells and IPEC-J2 cells were pre-treated with 200 nM and 400 nM Baf A1 at 37 °C for 1 h and then incubated with GDS01 or GDS09 strains for 1 h. DMSO was used as a negative control. The cells were collected 6 hpi and 9 hpi for qRT-PCR and Western blotting assay, respectively, to test the invasion efficiency of PEDV. **0.05 < P < 0.01; ***0.01 < P < 0.001; ****P < 0.001.

Figure 8

PEDV traffics to lysosome via endosomes. A, B Vero cells and IPEC-J2 cells were transfected with siRab7 and siVPS39 twice, respectively, and then infected with PEDV strains at 24 h after the second transfection. The cells were collected at 6 hpi for qRT-PCR analysis. Ctrl means control. C–E The Vero cells and IPEC-J2 cells were pre-cooled at 4 °C for 15 min, incubated with PEDV strains at 4 °C for 1 h, shifted to 37 °C to initiate internalization. The non-internalized viral particles were removed by washing. 30 min after shifting, the cells were fixed and stained with anti-PEDV-S (red) and anti-EEA1 (green) primary antibodies (C). 40 min after shifting, the cells were fixed and stained with anti-PEDV-S (red) and anti-Rab7 (green) primary antibodies (D). 50 min after shifting, the cells were fixed and stained with anti-PEDV-S (red) and anti-LAMP1 (green) primary antibodies (E). Scale bars indicate 5 μm in C–E. *P < 0.05; **0.05 < P < 0.01; ***0.01 < P < 0.001; ****P < 0.001.