Porcine Deltacoronavirus (PDCoV) Entry into PK-15 Cells by Caveolae-Mediated Endocytosis

Li, Shiqian; Xiao, Dai; Zhao, Yujia; Zhang, Luwen; Chen, Rui; Liu, Weizhe; Wen, Yimin; Liao, Yijie; Wu, Rui; Han, Xinfeng; Zhao, Qin; Du, Senyan; Yan, Qigui; Wen, Xintian; Cao, Sanjie; Huang, Xiaobo

doi:10.3390/v14030496

Cited by 7 publications

(12 citation statements)

References 45 publications

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“…Cholesterol plays an essential role in PDCoV entry into cells [ 19 , 41 ]. It could therefore be expected that statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, a key enzyme in cholesterol synthesis, reduce serum or cellular cholesterol levels, possibly inhibiting replication of coronaviruses, including PDCoV.…”

Section: Discussionmentioning

confidence: 99%

Detection of Porcine Deltacoronavirus RNA in the Upper and Lower Respiratory Tract and Biliary Fluid and the Effect of Infection on Serum Cholesterol Levels and Blood T Cell Population Frequencies in Gnotobiotic Piglets

Bedsted

Jung

Saif

2023

Veterinary Sciences

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Porcine deltacoronavirus (PDCoV) was first identified approximately a decade ago, but much is still obscure in terms of its pathogenesis. We aimed to further characterize PDCoV infection by investigating the presence of virus in respiratory and biliary tissues or fluids; T cell population frequencies in blood; and altered serum cholesterol levels. Twelve, 6-day-old, gnotobiotic piglets were inoculated oronasally with PDCoV OH-FD22 (2.6 × 107 FFU/pig). Six control piglets were not inoculated. Rectal swab (RS), nasal swab (NS), nasal wash (NW), bronchoalveolar lavage (BAL), and biliary fluid (BF) samples were collected at 2, 4, and 7 days post-inoculation (DPI) and tested for PDCoV RNA by RT-qPCR. Blood T cell populations and serum cholesterol levels were determined by flow cytometry and a colorimetric assay, respectively. Moderate to high, and low to moderate titers of PDCoV RNA were detected in RS and in NS, NW, BAL, and BF samples, respectively, of inoculated piglets. There were trends toward decreased CD4+CD8−, CD4−CD8+, and CD4+CD8+ blood T cell frequencies in inoculated piglets. Furthermore, serum cholesterol levels were increased in inoculated piglets. Overall, we found that PDCoV infection does not exclusively involve the intestine, since the respiratory and biliary systems and cholesterol metabolism also can be affected.

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Section: Discussionmentioning

confidence: 99%

Detection of Porcine Deltacoronavirus RNA in the Upper and Lower Respiratory Tract and Biliary Fluid and the Effect of Infection on Serum Cholesterol Levels and Blood T Cell Population Frequencies in Gnotobiotic Piglets

Bedsted

Jung

Saif

2023

Veterinary Sciences

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“…Endocytosis is mostly demonstrated by the addition of inhibitors. Common clathrin inhibitors include chlorpromazine and sucrose; common caveolin inhibitors include indomethacin, nystatin, methyl-β-cyclodextrin and genistein; common macropinocytosis pathway inhibitors include amiloride, cytochalasin and colchicine; and common forms of ATP energy suppression include sodium azide, 2-deoxyglucose and low temperature [40][41][42]. In this study, HCECs were co-incubated with chlorpromazine, indomethacin, amiloride, at 4 • C and with nanomicelles, separately, proving that the cellular uptake mechanism was energy-dependent, clathrin-and caveolin-mediated endocytosis.…”

Section: Discussionmentioning

confidence: 99%

Design of an L-Valine-Modified Nanomicelle-Based Drug Delivery System for Overcoming Ocular Surface Barriers

Cai

et al. 2022

Pharmaceutics

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The incidence of ocular surface disease (OSD) is increasing, with a trend towards younger ages. However, it is difficult for drugs to reach the deep layers of the cornea due to ocular surface barriers, and bioavailability is less than 5%. In this study, DSPE-PEG2000 was modified with L-valine (L-Val), and an HS15/DSPE-PEG2000-L-Val nanomicelle delivery system containing baicalin (BC) (BC@HS15/DSPE-PEG2000-L-Val) was constructed using thin-film hydration, with a high encapsulation rate, small particle size and no irritation to the ocular surface. Retention experiments on the ocular surface of rabbits and an in vivo corneal permeation test showed that, compared with the control, nanomicelles not only prolonged retention time but also enhanced the ability to deliver drugs to the deep layers of the cornea. The results of a protein inhibition and protein expression assay showed that nanomicelles could increase uptake in human corneal epithelial cells (HCEC) through energy-dependent endocytosis mediated by clathrin, caveolin and the carrier pathway mediated by PepT1 by inhibiting the overexpression of claudin-1 and ZO-1 and suppressing the expression of PepT1-induced by drug stimulation. These results indicate that BC@HS15/DSPE-PEG2000-L-Val is suitable for drug delivery to the deep layers of the ocular surface, providing a potential approach for the development of ocular drug delivery systems.

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“…Cell viability was assayed as described previously ( 20 ). Briefly, ST cells were seeded into 96-well plates and treated with different concentrations of inhibitors (100 μL) for 2 h at 37°C; after two washes with phosphate buffer solution (PBS), 10 µL of cell counting kit-8 (CCK-8) solution was added to 90 µL of DMEM in each well and incubated for 1 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Porcine deltacoronavirus enters porcine IPI-2I intestinal epithelial cells via macropinocytosis and clathrin-mediated endocytosis dependent on pH and dynamin ( 19 ). Our previous work also found that PDCoV enters PK-15 cells through caveolae-mediated endocytosis (CavME) ( 20 ).…”

Section: Introductionmentioning

confidence: 91%

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

Li,

Xiao,

Zhang

et al. 2023

Microbiol Spectr

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Porcine deltacoronavirus (PDCoV) is a newly emerged enteric pathogen that causes diarrhea, dehydration, vomiting, and in suckling piglets, a high rate of death. Here, we used endocytosis inhibitors, dominant-negative mutants, and small interfering RNA (siRNA) interference to investigate PDCoV entry into swine testicle (ST) cells. Results showed that chlorpromazine, dynasore, and methyl-β-cyclodextrin (MβCD) inhibited PDCoV internalization; immunofluorescence assay (IFA) of dominant-negative (DN) mutants and siRNA interference further demonstrated these results, indicating that PDCoV utilizes clathrin-mediated endocytosis and requires dynamin and cholesterol to enter ST cells. The role of endosomes in PDCoV infection was also investigated and we found that PDCoV does not require Rab5, Rab7, or Rab11 for productive infection, indicating that the transport of PDCoV in ST cells does not require the participation of the endosomal system. Our findings will provide additional understanding of the entry mechanisms of PDCoV and possible antiviral targets. IMPORTANCE 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.

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Porcine Deltacoronavirus (PDCoV) Entry into PK-15 Cells by Caveolae-Mediated Endocytosis

Cited by 7 publications

References 45 publications

Detection of Porcine Deltacoronavirus RNA in the Upper and Lower Respiratory Tract and Biliary Fluid and the Effect of Infection on Serum Cholesterol Levels and Blood T Cell Population Frequencies in Gnotobiotic Piglets

Detection of Porcine Deltacoronavirus RNA in the Upper and Lower Respiratory Tract and Biliary Fluid and the Effect of Infection on Serum Cholesterol Levels and Blood T Cell Population Frequencies in Gnotobiotic Piglets

Design of an L-Valine-Modified Nanomicelle-Based Drug Delivery System for Overcoming Ocular Surface Barriers

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

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