FSTL1 and TLR4 interact with PEDV structural proteins to promote virus adsorption to host cells

Abstract

Infection with porcine epidemic diarrhea virus (PEDV) results in enormous economic damage to the global swine industry. PEDV starts its life cycle by binding to the receptors of host cells and adsorbing onto the cellular surfaces. However, it is still unknown how PEDV adsorbs onto the surface of host cells and the mechanism beneath the interplay of host cell transmembrane protein with PEDV proteins. FSTL1, which is a secreted glycoprotein, participates in diverse pathological and physiological processes, including immune modulation and cell proliferation and differentiation. The transmembrane protein, TLR4, serves as a pattern recognition receptor recognizing a broad spectrum of pathogens, which exerts a crucial effect on the host immune system. In this study, we identified that FSTL1 promoted PEDV infection. Further studies demonstrated the interactive relationship between FSTL1 and PEDV structural proteins (N and S2). In addition, we also confirmed that TLR4 interacted with FSTL1 and PEDV N, S1, and S2 proteins on the cell surface. Moreover, FSTL1 promoted the interaction of TLR4 and PEDV and induced viral adsorption to host cells. This study offers explicit evidence that FSTL1 and TLR4 act as mediators for host cell adsorption of PEDV by interacting with PEDV N/S proteins.IMPORTANCEAs a highly infectious porcine epidemic diarrhea virus (PEDV)-induced intestinal condition of swine, porcine epidemic diarrhea (PED) results in a 100% death rate among suckling piglets and poses a serious economic burden to global swine farming. Therefore, it is essential to investigate the mechanism of virus infection, replication, and proliferation. Virus begins its life cycle by binding to the receptor of host cells and adsorbing onto the cellular surfaces. However, it remains unclear how PEDV adsorbs onto the host cell surfaces. This study revealed that host protein FSTL1 interacted with the PEDV N and S2 proteins, while TLR4 interacted with the FSTL1 and PEDV proteins (N, S1, and S2). Moreover, we thoroughly and methodically demonstrated that FSTL1 was engaged in the PEDV internalization and attachment processes by promoting the recognition of PEDV N\S proteins by TLR4 and induced the viral adsorption to host cells.

Keywords: FSTL1; PEDV; TLR4; structural proteins; virus adsorption.

Fig 1

FSTL1 was upregulated by the PEDV nsp7 protein through transcription factor KLF5. (A and B) PEDV-infected or negatively infected LLC-PK1 cells (MOI = 1) were explored by western blot and qRT-PCR. (C and D) FSTL1 expression was determined through western blot and qRT-PCR in gradient PEDV-infected LLC-PK1 cells. (E) LLC-PK1 cells were transfected with PEDV structural and non-structural proteins, and then the mRNA expression levels of FSTL1 were identified by qRT-PCR. (F) Western blot was used to detect the expression levels of endogenous FSTL1 in nsp7-transfected LLC-PK1 cells. (G) HEK 293T cells transfected with pGL3-basic luciferase vector carrying truncated constructs (−2,000 to −1) of the FSTL1 promoter were explored for the luciferase activity. (H) The TFBS of the FSTL1 promoter was expected with JASPAR. (I) Relative mRNA levels of predicted genes investigated through qRT-PCR in LLC-PK1 cells infected with PEDV. (J) The luciferase activity was explored in MEIS1, KLF5, ID4, Pax2, and E2F4 over-expressing HEK 293T cells. (K) LLC-PK1 cells were transfected with FLAG-KLF5 plasmid and collected to perform a ChIP assay. Data are shown to be means ± SD of triplicate samples. *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed Student’s t-test).

Fig 2

FSTL1 promotes PEDV replication. (A–C) LLC-PK1 cells were transfected with FLAG-FSTL1 plasmids and infected with PEDV at an MOI of 0.01. The PEDV titers were analyzed using western blot, qRT-PCR, and TCID₅₀. (D and E) pLV-Vero and pLV-FSTL1-Vero cells were infected with PEDV at an MOI of 0.01. The PEDV titers were investigated with western blot and qRT-PCR. (F and G) LLC-PK1 cells were transfected with the gradient concentration of FSTL1 plasmids and infected with PEDV at an MOI of 0.01. The PEDV titers were explored using western blot and qRT-PCR. (H and I) LLC-PK1 cells were transfected with FSTL1 siRNA and infected with PEDV at the MOI of 0.01. The cell lysates were determined through western blot and qRT-PCR. Data are indicated as means ± SD of triplicate samples. *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed Student’s t-test).

Fig 3

FSTL1 interacts with PEDV N and S2 proteins. (A and C) HEK 293T cells subject to transfection with the FLAG-FSTL1 and HA-N or HA-S2 plasmids. The cells were assayed by Co-IP with anti-Flag binding beads. Western blot was used to explore the precipitated proteins. (B and D) FSTL1, PEDV N, and PEDV S2 were cloned into pCold-TF or pCold-GST plasmids and denoted into BL21 (DE3) bacterial strain for affinity isolation of the GST pull-down assay. The eluted proteins were explored using western blot. (E) HeLa cells were transfected with FLAG-FSTL1 and HA-N or HA-S2 plasmids. The cells were labeled with specific antibodies. DAPI (4,6-diamidino-2-phenylindole) labeling for the cellular nuclei and fluorescent signals were monitored using a confocal immunofluorescent microscope (scale bars = 100 µm).

Fig 4

FSTL1 facilitates PEDV virion’s attachment to the cell membrane. (A and B) LLC-PK1 cells were transfected with FLAG-FSTL1 plasmids, infected with PEDV (MOI = 1) for 2 h at 4°C, and rinsed thrice with pre-cooled PBS to perform a virus adsorption assay. The PEDV titers were investigated using western blot and qRT-PCR. (C and D) LLC-PK1 cells were exposed to transfection with FSTL1 siRNA or siNC and infected with PEDV at an MOI of 1 at 4°C. The PEDV titer was tested using western blot and qRT-PCR. (E and F) The virus adsorption assay was carried out with FSTL-overexpressing cell lines, and the titers of PEDV were tested through qRT-PCR and western blot. (G) LLC-PK1 cells were pretreated with purified FSTL1 proteins at a concentration of 120 μg/mL and seeded with PEDV (MOI = 1) to conduct a virus adsorption assay. Western blotting was used to test the PEDV titers. (H and I) Detecting the attachment of viral particles based on qRT-PCR and western blotting after 1 μg of FSTL1 antibody occlusion in LLC-PK1 cells.

Fig 5

TLR4 interacts with PEDV N, S1, and S2 proteins. (A) HEK 293T cells were transfected with FLAG-FSTL1 and MYC-TLR4 plasmids. Co-IP assay was used for the analysis of the protein interaction. (B–D) HEK 293T cells were transfected with FLAG-TLR4 and HA-N, HA-S1, or HA-S2 plasmids. Co-IP assay was employed to explore the protein interaction. (E) HeLa cells were transfected with indicated plasmids. The cells were labeled with specific antibodies and monitored the fluorescent signals with a confocal immunofluorescent microscope (scale bars = 100 µm).

Fig 6

FSTL1 promotes the interaction of TLR4 and PEDV N/S. (A and B) LLC-PK1 cells were transfected with TLR4 siRNA and FLAG-FSTL1 plasmid, followed by infection with PEDV (MOI = 1) at 4°C for 2 h. The PEDV titers were determined by qRT-PCR and western blot. (C and D) LLC-PK1 cells were transfected with FSTL1 and TLR4 plasmids and infected with PEDV (MOI = 1) at 4°C for 2 h. The PEDV titers were detected using qRT-PCR and western blot. (E and F) HEK 293T cells were transfected with FLAG-TLR4, MYC-FSTL1, and HA-N or HA-S2 plasmids. Co-IP assay was used for exploring the protein interaction.

Fig 7

FSTL1 promotes attachment of PEDV by interacting with the TLR4 proteins. FSTL1 and TLR4 interact at the surface of the cell membrane. FSTL1 recognizes and interacts with PEDV virions that are free from the cell membrane. Then, FSTL1 promotes the interaction of PEDV virions and TLR4. Finally, FSTL1, TLR4, and PEDV virions form a complex that promotes the attachment of virions to the cell membrane.