Research Center for Swine Diseases, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China.
Sichuan Science-Observation Experimental Station for Veterinary Drugs and Veterinary Diagnostic Technology, Ministry of Agriculture, Chengdu 611130, China.
Viruses. 2022 Feb 28;14(3):496. doi: 10.3390/v14030496.
(1) Background: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus affecting pig breeding industries worldwide, and its pathogenic mechanism remains unclear. (2) Methods: In this study, we preliminarily identified the endocytic pathway of PDCoV in PK-15 cells, using six chemical inhibitors (targeting clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis pathway and endosomal acidification), overexpression of dominant-negative (DN) mutants to treat PK-15 cells and proteins knockdown. (3) Results: The results revealed that PDCoV entry was not affected after treatment with chlorpromazine (CPZ), 5-(N-ethyl-N-isopropyl) amiloride (EIPA)or ammonium chloride (NHCl), indicating that the entry of PDCoV into PK-15 cells were clathrin-, micropinocytosis-, PH-independent endocytosis. Conversely, PDCoV infection was sensitive to nystatin, dynasore and methyl-β-cyclodextrin (MβCD) with reduced PDCoV internalization, indicating that entry of PDCoV into PK-15 cells was caveolae-mediated endocytosis that required dynamin and cholesterol; indirect immunofluorescence and shRNA interference further validated these results. (4) Conclusions: In conclusion, PDCoV entry into PK-15 cells depends on caveolae-mediated endocytosis, which requires cholesterol and dynamin. Our finding is the first initial identification of the endocytic pathway of PDCoV in PK-15 cells, providing a theoretical basis for an in-depth understanding of the pathogenic mechanism of PDCoV and the design of new antiviral targets.
(1) 背景：猪德尔塔冠状病毒（PDCoV）是一种新出现的肠道病毒，影响着全球的养猪业，但其致病机制尚不清楚。(2) 方法：本研究初步鉴定了 PDCoV 在 PK-15 细胞中的内吞途径，使用了六种化学抑制剂（针对网格蛋白介导的内吞作用、小窝介导的内吞作用、巨胞饮途径和内体酸化）、过表达显性负（DN）突变体处理 PK-15 细胞和蛋白敲低。(3) 结果：结果表明，在用氯丙嗪（CPZ）、5-（N-乙基-N-异丙基）阿米洛利（EIPA）或氯化铵（NHCl）处理后，PDCoV 的进入不受影响，表明 PDCoV 进入 PK-15 细胞的过程不受网格蛋白、微胞饮、pH 无关内吞作用的影响。相反，PDCoV 感染对制霉菌素、dynasore 和甲基-β-环糊精（MβCD）敏感，PDCoV 内化减少，表明 PDCoV 进入 PK-15 细胞是网格蛋白依赖的内吞作用，需要 dynamin 和胆固醇；间接免疫荧光和 shRNA 干扰进一步验证了这些结果。(4) 结论：综上所述，PDCoV 进入 PK-15 细胞依赖于小窝介导的内吞作用，需要胆固醇和 dynamin。我们的发现首次初步鉴定了 PDCoV 在 PK-15 细胞中的内吞途径，为深入了解 PDCoV 的致病机制和设计新的抗病毒靶点提供了理论依据。
