Porcine deltacoronavirus enters ST cells by clathrin-mediated endocytosis and does not require Rab5, Rab7, or Rab11

Abstract

Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus threatening pig industries worldwide. Our previous work showed that PDCoV enters porcine kidney (PK-15) cells through a caveolae-dependent pathway, but the entry mechanism for PDCoV into swine testicle (ST) cells remains unclear. Mechanisms of virus entry can be different with different virus isolates and cell types. Here, we determined that PDCoV enters ST cells via clathrin-mediated endocytosis. Additionally, we found that PDCoV entry does not require Rab5, Rab7, or Rab11. These findings provide additional understanding of the entry mechanisms of PDCoV and possible antiviral targets.

Keywords: Rab proteins; clathrin; endocytosis; porcine deltacoronavirus (PDCoV).

Fig 1

Cell viability. ST cells were seeded into 96-well plates at 3 × 10⁴ cells/well, incubated for 24 h and treated with inhibitors or left untreated for 2 h. After two washes with PBS, 10 µL of CCK-8 solution was added to each well and incubated for 1 h at 37°C. Absorbance was measured at 450 nm. The bars indicate the mean ±  SD from three independent experiments. (A) Nystatin, (B) MβCD, (C) dynasore, (D) chloroquine, (E) EIPA, and (F) NH₄Cl.

Fig 2

PDCoV entry into ST cells depends on clathrin-mediated endocytosis. (A) ST cells were treated with CPZ for 1 h at 37°C, then incubated with PDCoV (MOI = 5) for 1 h at 4°C (binding step), and then shifted to 37°C for 1 h (entry step). qRT-PCR of cell lysates was done to determine viral mRNA levels. (B–D) Cells were mock-treated or treated with CPZ for 1 h followed by infection with PDCoV (MOI = 0.1). At 24 hpi, cell lysates were used for (B) qRT-PCR to determine viral mRNA levels, (C) Western blotting to determine intracellular N protein levels, and (D) TCID₅₀. (E) ST cells were transfected plasmids expressing with WT or DN EPS15 for 24 h, followed by infection with PDCoV (MOI = 0.1) for 6 h, then fixed and immuno-stained. The magnification is 80×. (F–I) Clathrin knockdown and normal cells were infected with PDCoV (MOI = 0.1) for 24 h. (F) The knockdown efficiency was determined by Western blotting. PDCoV (G) N protein levels, (H) TCID₅₀, and (I) N mRNA levels were determined. *P < 0.05, **P < 0.01, ***P < 0.001, and ns: no significant difference.

Fig 3

PDCoV entry is caveola independent. (A) ST cells were pretreated with nystatin for 1 h at 37°C, then incubated with PDCoV (MOI = 5) for 1 h at 4°C (binding step), and then shifted to 37°C for 1 h (entry step). Viral N mRNA was quantitated by qRT-PCR. (B–D) Cells were mock-treated or treated with nystatin for 1 h followed by infection with PDCoV (MOI = 0.1) for 24 h. (B) N mRNA levels were quantitated by qRT-PCR. (C) Intracellular N protein levels were detected by Western blot. (D) Viral titers were determined by TCID₅₀. (E) ST cells were transfected with caveolin-1 WT or DN expressing plasmids for 24 h, followed by PDCoV infection (MOI = 0.1) for 6 h. Cells were fixed and immuno-stained. The magnification is 80×. (F–I) Caveolin-1 knockdown and normal cells were infected with PDCoV (MOI = 0.1). (F) Knockdown efficiency was determined by qRT-PCR. At 24 hpi, (G) N mRNA levels, (H) viral titers, and (I) N protein levels were determined. *P < 0.05, **P < 0.01, ***P < 0.001, and ns: no significant difference.

Fig 4

PDCoV entry into ST cells is independent of macropinocytosis. (A) ST cells were pretreated with EIPA for 1 h at 37°C, then incubated with PDCoV (MOI = 5) for 1 h at 4°C (binding step), and then shifted to 37°C for 1 h (entry step). Viral N mRNA was quantitated by qRT-PCR. (B–D) Cells were mock-treated or treated with EIPA for 1 h followed by infection with PDCoV (MOI = 0.1) for 24 h. (B) N mRNA levels were quantitated by qRT-PCR. (C) Intracellular N protein levels were detected by Western blot. (D) Viral titers were determined by TCID₅₀. ns: no significant difference.

Fig 5

PDCoV entry into ST cells does not require low pH. (A) ST cells were pretreated with NH₄Cl at 37°C for 1 h and infected with PDCoV (MOI = 5) at 4°C for 1 h (binding step), and then shifted to 37°C for 1 h (entry step). Viral N mRNA was quantitated by qRT-PCR. (B–D) Cells were mock-treated or treated with NH₄Cl for 1 h followed by infection with PDCoV (MOI = 0.1) for 24 h. (B) N mRNA levels were quantitated qRT-PCR. (C) Intracellular N protein levels were detected by Western blot. (D) Viral titers were determined by TCID₅₀. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 6

PDCoV entry into ST cells is dynamin dependent. (A) ST cells were pretreated with dynasore for 1 h at 37°C, then incubated with PDCoV (MOI = 5) for 1 h at 4°C (binding step), and then shifted to 37°C for 1 h (entry step). Viral N mRNA was quantitated by qRT-PCR. (B–D) Cells were mock-treated or treated with dynasore for 1 h followed by infection with PDCoV (MOI = 0.1) for 24 h. (B) N mRNA levels were quantitated by qRT-PCR. (C) Intracellular N protein levels were detected by Western blot. (D) Viral titers were determined by TCID₅₀. (E) ST cells were transfected with dynamin-2 WT or DN expressing plasmids for 24 h, followed by infection with PDCoV (MOI = 0.1) for 6 h. Cells were fixed and immuno-stained. The magnification is 80×. (F–I) Dynamin-2 knockdown and normal cells were infected with PDCoV (MOI = 0.1). The knockdown efficiency was verified by qRT-PCR (F). At 24 hpi, the mRNA level of PDCoV, virus titer, and N protein level were determined by qRT-PCR (G), TCID50 (H), and Western blotting (I), respectively. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 7

PDCoV entry into ST cells depends on cholesterol. (A) ST cells were pretreated with MβCD for 1 h at 37°C, then incubated with PDCoV (MOI = 5) for 1 h at 4°C (binding step), and then shifted to 37°C for 1 h (entry step). Viral N mRNA was quantitated by qRT-PCR. (B–D) Cells were mock-treated or treated with MβCD for 1 h followed by infection with PDCoV (MOI = 0.1) for 24 h. (B) N mRNA levels were quantitated by qRT-PCR. (C) Intracellular N protein levels were detected by Western blot. (D) Viral titers were determined by TCID₅₀. ns: no significant difference.

Fig 8

Levels of Rabs in PDCoV infected on ST cells. ST monolayers were incubated with PDCoV (MOI = 0.1) for 1.5 h at 37°C. Samples were collected for Western blotting analysis at the indicated times post-infection.

Fig 9

Effect of Rab5 WT overexpression and DN expression on PDCoV infection. ST cells were transfected with Rab5 WT or DN expressing plasmids for 24 h, followed by infection with PDCoV (MOI = 0.1) for 6 h. Cells were fixed and immuno-stained. The magnification is 80×.

Fig 10

Effect of Rab7 on PDCoV infection. (A) ST cells were transfected with Rab7 WT or DN expressing plasmids for 24 h, followed by infection with PDCoV (MOI = 0.1) for 6 h. Cells were fixed and immuno-stained. The magnification is 80×. (B) Knockdown efficiency was verified by Western blotting. (C–E) Rab7 knockdown and normal cells were infected with PDCoV (MOI = 0.1) for 24 h. N mRNA levels, viral titer, and N protein levels were determined by (C) qRT-PCR, (D) TCID₅₀, and (E) Western blotting. ns: no significant difference.

Fig 11

Effect of Rab11 on PDCoV infection. (A) ST cells were transfected with WT or DN expressing Rab11 expressing plasmids for 24 h, followed by infection with PDCoV (MOI = 0.1) for 6 h. Cells were fixed and immuno-stained. The magnification is 80×. (B) Knockdown efficiency was verified by Western blotting. (C–E) Rab11 knockdown and normal cells were infected with PDCoV (MOI = 0.1) for 24 h. N mRNA levels, viral titer, and N protein levels were determined by (C) qRT-PCR, (D) TCID₅₀, and (E) Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001, and ns: no significant difference.