Citation
- Authors: Xu W. et al.
- Year: 2022
- Journal: Int J Biochem Cell Biol 153 106325
- Applications: in vitro / DNA / PEIpro
- Cell types:
- Name: BHK
- Name: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
Method
Subcellular localization
BHK cells were seeded on 12 well glass bottom plates (Cellvis, USA), 3 × 105 lives cells/well. Overnight, cells were transfected with a total of 3 µg of different plasmids using PEIpro (Polyplus, France) according to the manufacturer's protocol. Forty-eight hours after transfection, cells were washed with PBS and fixed in 4% polyoxymethylene (Beyotime, China) for 10 min at room temperature, then stained the nucleus with DAPI. A laser confocal microscope were used to take pictures, which then were analyzed with ImageJ software.
Co-IP and western blot
293 T cells were seeded in six-well plates. Overnight, cells were transfected with a total of 4 µg of various plasmids using PEIpro. Forty-eight hours later, the cells were collected to a centrifuge tube and centrifuged at 5,000 rpm for 5 min at 4 ℃.
Abstract
IFITM proteins are a host restriction factor with broad-spectrum antiviral activity, but the role in the paramyxovirus entry remains unclear. Nipah virus (NiV) is a zoonotic virus of the paramyxoviridae with extremely high lethality. Here, we assessed the role of IFITM3 on NiV G and F glycoprotein-mediated virus entry. Using NiV pseudovirus bearing NiV G and F proteins to infect IFITM3-induced MDCK cells, we found that overexpression of IFITM3 promotes NiV G and F proteins-mediated virus entry. Mechanistically, the subcellular distribution showed that F protein completely co-localized with IFITM3, but G protein does not. Immunoprecipitation further indicated that IFITM3 strongly captures F protein rather than G protein. F protein truncation found that the F1 subunit completely co-localized and captures with IFITM3, but not the F2 subunit. Furthermore, IFITM3 strongly binds to F1 truncations containing fusion peptide (FP), and F1 strongly captures IFITM3 truncation with the intramembrane domain (IMD). Together, the results suggest that IFITM3 can promote NiV G and F proteins-mediated virus entry into MDCK cells, and IFITM3 directly interacts with the F1 subunit of NiV F protein dependent on the former's IMD and the latter's FP, which may occur after incorporation of fusion peptides into the cell membrane following virus fusion activation.