Knockout of CLTC gene reduces but not completely block SFTSV infection.

Tiezhu Liu, Jiajia Li, Xueqi Wang, Tao Huang, Wei Wu, Aqian Li, Chuan Li, Xiaoxia Huang, Qin Wang, Dexin Li, Shiwen Wang, Mifang Liang

PloS one 2023

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Clathrin is a key protein for viruses to enter host cells. Previous studies often use clathrin inhibitors or gene knockdown technology to partially inhibit the function of clathrin, but whether SFTSV can infect host cells without clathrin expression remains unclear. In this research, a clathrin heavy chains (CLTC) knockout A549 cell line was established by CRISPR/Cas9 technology, and the knockout of CLTC was verified by PCR, Western blot, immunofluorescence and T7E1 analysis. The off-target effect was evaluated by PCR combined with Sanger sequencing. Furthermore, this research verified that SFTSV infection was significantly inhibited, but not completely blocked, due to the deletion of CLTC protein. Our research also found that lipid raft inhibitor Filipin, other than macropinocytosis inhibitor EIPA, could significantly reduce SFTSV infection, and the inhibition was more obviously observed when Filipin was used in CLTC knockout cells. These result indicated that clathrin-dependent and lipid raft mediated endocytosis are the major two mode used by SFTSV entry. In conclusion, this study constructed a CLTC knockout cell line, which, for the first time, established a cell model for the study of the function of CLTC protein, and provided direct evidence that SFTSV pendent could still infect cells without clathrin. Additionally, we confirmed that lipid raft mediated endocytosis, as a clathrin-independent pathway, could be another key mode for SFTSV entry. Copyright: © 2023 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Tiezhu Liu, Jiajia Li, Xueqi Wang, Tao Huang, Wei Wu, Aqian Li, Chuan Li, Xiaoxia Huang, Qin Wang, Dexin Li, Shiwen Wang, Mifang Liang. Knockout of CLTC gene reduces but not completely block SFTSV infection. PloS one. 2023;18(8):e0285673