Fig 1.
FUT8, but not other FUT family members, is a host factor essential for TGEV replication.
(A) Genomic sequence analysis of FUT8 in WT cells and FUT8-KO cells. (B) Western blot analysis validated the protein expression levels of endogenous FUT8 in FUT8 KO and WT cells. GAPDH was used as an internal control. (C) Cell viability in FUT8 KO and WT PK-15 cells was measured by MTS assay. (D) Immunofluorescence assays were used to detect TGEV N protein expression in FUT8 KO and WT cells infected with TGEV at different MOIs (MOI = 0.01 and 1) at 24 hpi. Scale bar, 200 µm. (E) TGEV titers were measured at 12, 24, 36, and 48 hpi. (F) Western blot assay was used to detect TGEV N protein expressed in FUT8 KO and WT cells following infection with TGEV (MOI = 1) at 24 hpi. GAPDH was used as an internal control gene. (G) TGEV titers were measured at 24 hours at ST-WT and ST-FUT8-KO cells. (H) Immunofluorescence assays were used to detect TGEV N protein expression in FUT 1-13 (except FUT6 and FUT8) KO and WT cells infected with TGEV (MOI = 0.01) at 24 hpi. Scale bar, 400 µm. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. P values were determined by two-sided Student’s t-test. Data are representative of at least three independent experiments.
Fig 2.
FUT8 regulates TGEV infection dependent on its glycosyltransferase activity.
(A) A schematic diagram of FUT8 enzyme active site mutations is shown. (B) Rescue assays were performed for FUT8 KO, FUT8-KO-rescue-WT, and FUT8-KO-rescue-R365A cells infected with TGEV. Expression of WT and R365A FUT8 (left) and TGEV N gene (right) was measured in FUT8 KO cells.(C and E) PK-15 cells were treated with different concentrations (200, 400, and 800 µM) of 2F-Peracetyl-Fucose for 5 days, and cells were infected with TGEV (MOI = 0.1). TGEV titers (E) were measured at 24 hpi, and TGEV N protein expression was detected by immunofluorescence assay (D). (D and F) PK-15 cells were treated with different concentrations (50 and 100 µM) of FDW028 for 2 days, and cells were infected with TGEV (MOI = 0.01). TGEV titers (D) were measured at 24 hpi, and TGEV N protein expression was detected by immunofluorescence assay (F). Scale bar, 400 µm. **p < 0.01; ***p < 0.001; ****p < 0.0001. p values were determined by two-sided Student’s t-test. Data are representative of at least three independent experiments.
Fig 3.
FUT8 KO inhibits viral entry by regulating the core fucosylation of APN.
(A) TEM was used to evaluate the effects of FUT8 KO on virus particle assembly. Compared to WT cells, which contained numerous virions (blue arrows), almost no virus-like particles wrapped in vesicles of varying sizes were observed in FUT8 KO cells. Scale bars, 500 nm. Mock, uninfected cells; ER, endoplasmic reticulum; N, nucleus; double membrane vesicles (DMV) are indicated by a red asterisk. (B) IFAs were used to evaluate early-stage TGEV replication by detecting dsRNA formation in WT and FUT8 KO cells infected with TGEV (MOI = 1) at 6 hpi. (C) TGEV-BAC plasmids were transfected into pAPN KO cells and pAPN KO + FUT8 KO cells. After 60 hours, cells were harvested, and RT-qPCR was performed to detect TGEV N gene copy number. (D) WT, FUT8 KO, and pAPN KO cells were infected with TGEV (MOI = 5) at 4 °C for 1 hour, and TGEV adsorption was assessed. Cells were harvested, and viral RNA was extracted to determine virion attachment to the cell surface. (E) WT, FUT8 KO, and pAPN KO cells were infected with TGEV (MOI = 5) at 4 °C for 1 hour, followed by incubation at 37 °C for 30 minutes. Cells were treated with cold acidic PBS-HCl (pH = 3.0) to remove attached but un-internalized viral particles. IThe internalization of TGEV was evaluated by RT-qPCR. (F) A membrane fusion assay assessed the fusion ability of the TGEV spike protein in WT cells and FUT8 KO PK-15 cells. (G) PK-15 cells were treated with different concentrations (50, 200, 400, and 800 µM) of 2F-Peracetyl-Fucose for 5 days, and cells were incubated with TGEV (MOI = 5) to detect TGEV adsorption and internalization by RT-qPCR. (H) A membrane fusion assay assessed the fusion ability of the TGEV spike protein in pAPN overexpressed in WT and FUT8 KO 293T cells. (I) mRNA and protein expression levels of pAPN were measured in WT and FUT8 KO 293T cells. (J) Inhibitor membrane fusion assay, 293T cells were transfected with pAPN for 8 hours and treated with 20, 40, and 80 μg/mL AOL for 16 hours, followed by a cell membrane fusion assay. *p < 0.1;**p < 0.01; ***p < 0.001; ****p < 0.0001. p values were determined by two-sided Student’s t-test. Data representat at least three independent experiments.
Fig 4.
Core fucosylation of pAPN N736 by FUT8 is important for TGEV entry.
(A) The SPR binding assays using Fc-tagged TGEV RBD and pAPN proteins purified separately from WT and FUT8-KO 293F cells. (B) Structural analysis of the RBD (medium slate blue) interacting with pAPN (lime green) using ChimeraX. The first N-linked glycan (N-acetyl glucosamine) on pAPN Asn736 is shown (a transparent surface), interacting with the RBD (Protein Data Bank accession code 4F5C). (C) Interaction analysis of pAPN and RBD via Ligplot. (D) Flow cytometry analysis of TGEV-S protein binding to pAPN or pAPN N736A in 293T or 293T-FUT8 KO cells. (E) Schematic of the pAPN or pAPN N736A membrane fusion assay. The illustrations were created using Figdraw: https://www.figdraw.com. Copyright ID:YTOIPf8300. (F) Membrane fusion assay assessing TGEV spike protein fusion in pAPN or pAPN N736A-expressing WT and FUT8 KO 293T cells. ****p < 0.0001. p values were determined using a two-sided Student’s t-test. Data represent at least three independent experiments.
Fig 5.
Glycosylation profile on pAPN characterized.
(A) Mapping of core fucosylation sites on pAPN. Six pAPN N-glycosylation sites (N82, N124, N556, N569, N646, and N736) are core-fucosylated. (B) The intensity of core fucosylation on pAPN N736 site of WT and FUT8-KO cells. The “intensity of core fucosylation” refers to the total MS1 signal intensity of all identified core fucosylated glycopeptides derived from LC-MS/MS analysis. Detailed glycan analysis the relative ratio of pAPN N736 site in WT (C) and FUT8-KO cells (D).The complex type glycans were observed in all N-glycosylation sites(C).Glycan species are labeled using GT identifiers for simplicity, the detailed glycan structure corresponding to each GT number are provided in S1 Table.
Fig 6.
FUT8 is a key entry factor for multiple α-CoVs.
(A) Sequence alignment of homologous pig APN, canine APN, feline APN, and human APN. pAPN, cAPN, and fAPN share a conserved “NWT” glycosylation motif at sites 736/747/740. (B) Structural analysis of the CCoV RBD (yellow) interacting with cAPN (hot pink) (PDB: 7U0L). The CCoV RBD Q545 side chain hydrogen bonds with the cAPN N747 side chain, the first N-linked NAG, and core fucose. (ChimeraX). (C) Membrane fusion assay assessing CCoV spike protein fusion in cAPN- or cAPN N747A-expressing WT and FUT8 KO 293T cells. (D) Structural analysis of the FCoV RBD (salmon) interacting with fAPN (magenta) (PDB: 9DAZ). (E) Membrane fusion assay assessed FIPV spike protein fusion in fAPN- or fAPN N740A-expressing WT and FUT8 KO 293T cells. (F) TCID50 assay quantifying FIPV titers in CRFK and CRFK-FUT8 KO cells 24 hours after infection (MOI = 0.1). (G) The diagram displayed FUT8 as a conserved host factor involved in multiple α-CoV infect by enabling core fucosylation of the receptor APN. The illustrations in this figure were created using Figdraw:https://www.figdraw.com. Copyright ID:RSOUAfaaa6. **p < 0.01; ***p < 0.001; ****p < 0.0001. p values were determined using a two-sided Student’s t-test. Data represent at least three independent experiments.