Mechanisms Underlying the Promotion of Porcine deltacoronavirus Infection in ST Cells by Tight Junction Protein Occludin
ZHANG Xue-Li1, XIA Liang-Xing1, ZHANG Shuai1,*, BAO Wen-Bin1,2,*
1 College of Animal Science and Technology/Jiangsu Key Laboratory of Animal Genetic Breeding and Molecular Design, Yangzhou University, Yangzhou 225009, China; 2 College of Animal Science and Technology, International Joint Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou 225009, China
Abstract：Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that causes acute watery diarrhea, vomiting, and dehydration in neonatal piglets (Sus scrofa). PDCoV infection leads to villus atrophy, crypt hyperplasia, and disruption of the intestinal barrier, resulting in high mortality and substantial economic losses to the swine industry. Occludin, a key component of tight junctions, plays critical roles in maintaining epithelial barrier integrity and mediating signal transduction. Using swine testicular (ST) cells as a model, the effect of PDCoV infection on Occludin expression was assessed by Western blot and qRT-PCR analyses in this study. Results demonstrated that both Occludin mRNA and protein levels decreased significantly over time post-infection (P<0.01), suggesting a correlation between PDCoV infection and Occludin downregulation. To further clarify the functional role of Occludin in viral replication, Occludin overexpressed or knocked down via plasmid overexpression and short hairpin RNA (shRNA) interference, respectively. The results showed that Occludin overexpression significantly promoted PDCoV infection (P<0.0001), while Occludin knockdown significantly inhibited PDCoV infection (P<0.0001), indicating that Occludin was a critical host factor involved in PDCoV replication. Furthermore, to explore the entry pathways through which Occludin promotes viral infection, cells were treated with the clathrin inhibitor Pitstop 2 and the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA). Both inhibitors markedly attenuated the Occludin overexpression-mediated enhancement of PDCoV infection. Collectively, The findings elucidated that Occludin positively regulated PDCoV entry via clathrin-mediated endocytosis and macropinocytosis, providing a theoretical basis for in-depth understanding of PDCoV pathogenesis and for developing novel host factor-targeted control strategies.
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