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Updated: Jul 18, 2025
Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis
Published on: January 30, 2020
Knockout of CLTC gene reduces but not completely block SFTSV infection
Tiezhu Liu1, Jiajia Li2, Xueqi Wang3
1National Health Commission Key Laboratory for Medical Virology, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
Abstract:
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.
Insights
SFTSV can still infect cells even without clathrin heavy chains (CLTC). Lipid raft-mediated endocytosis acts as a clathrin-independent pathway for SFTSV entry, alongside clathrin-dependent mechanisms.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Clathrin is crucial for viral entry into host cells.
- Previous studies partially inhibited clathrin function, leaving the role of complete clathrin absence in SFTSV infection unclear.
Purpose of the Study:
- To investigate SFTSV entry mechanisms in the absence of clathrin.
- To establish a clathrin-heavy-chain (CLTC) knockout cell line for studying clathrin function.
- To identify alternative viral entry pathways for SFTSV.
Main Methods:
- CRISPR/Cas9 technology was used to create a CLTC knockout A549 cell line.
- PCR, Western blot, immunofluorescence, and T7E1 analysis verified CLTC knockout.
- Off-target effects were assessed using PCR and Sanger sequencing.
- SFTSV infection assays were performed with and without CLTC, using lipid raft and macropinocytosis inhibitors.
Main Results:
- SFTSV infection was significantly reduced, but not abolished, in CLTC knockout cells.
- Lipid raft inhibitor Filipin, but not macropinocytosis inhibitor EIPA, reduced SFTSV infection.
- The inhibitory effect of Filipin was more pronounced in CLTC knockout cells.
- This suggests both clathrin-dependent and lipid raft-mediated endocytosis are major SFTSV entry routes.
Conclusions:
- A CLTC knockout cell line was successfully generated, providing a novel model for clathrin function studies.
- SFTSV can infect cells independently of clathrin, utilizing lipid raft-mediated endocytosis as a key alternative pathway.
