Jialin Zhang& , Jianfei Chen& , Da Shi, Hongyan Shi, Xin Zhang, Jianbo Liu, Liyan Cao, Xiangdong Zhu, Ye Liu, Xiaobo Wang, Zhaoyang Ji and Li Feng*
J Biol Chem. 2019 May 8. pii: jbc.RA119.007779. doi: 10.1074/jbc.RA119.007779
Porcine deltacoronavirus (PDCoV) is a pathogen belonging to the deltacoronavirus family that in 2014 caused outbreaks of piglet diarrhea in the United States. To identify suitable therapeutic targets, a more comprehensive understanding of the viral entry pathway is required, particularly of the role of proteases. Here, we identified the proteases that activate the viral spike (S) glycoprotein to initiate cell entry and also pinpointed the host-cellular pathways that PDCoV uses for entry. Our results revealed that cathepsin L (CTSL) and cathepsin B (CTSB) in lysosomes and extracellular trypsin in cell cultures independently activate the S protein for membrane fusion. Pre-treating the cells with the lysosomal acidification inhibitor bafilomycin-A1 (Baf-A1) completely inhibited PDCoV entry, and siRNA-mediated ablation of CTSL or CTSB expression significantly reduced viral infection, indicating that PDCoV uses an endosomal pathway for entry. Of note, trypsin treatment of cell cultures also activated PDCoV entry, even when the endosomal pathway was inhibited. This observation indicated that trypsin-induced S protein cleavage and activation in cell cultures enables viral entry directly from the cell surface. Our results provide critical insights into the PDCoV infection mechanism, uncovering two distinct viral entry pathways: one through cathepsin L and cathepsin B in the endosome and another via a protease at the cell surface. Since PDCoV infection sites represent a proteases-rich environment, these findings suggest that endosome inhibitor treatment alone is insufficient to block PDCoV entry into intestinal epithelial cells in vivo. Therefore, approaches that inhibit viral entry from the cell membrane should also be considered.
Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
protease; proteolytic enzyme; virology; virus; virus entry