CD1d facilitates African swine fever virus entry into the host cells via clathrin-mediated endocytosis

Figures

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  Figure 1.. ASFV p72 facilitates viral entry and interacts with CD1d protein. (A) Immunocytochemistry was performed to detect the co-localization of DiD-ASFV (Red) and p72 (Green) after virus infection of PAMs for 1 h at 4°C. As negative controls, co-localization of DiD-ASFV and p34 or pB318L were also detected. The cell membrane was stained with WGA (Grey). The nuclei were stained with DAPI (Blue). White arrows show the co-localized DiD-ASFV and p72. Bar = 5 μm. (B–E) ASFV virions were incubated with anti-p72, anti-p34 and anti-pB318L antibodies at the indicated concentrations for 4 h at 4°C. Then, PAMs were infected with 1 MOI rASFV-Gluc-GFP virions-antibody mixtures for 24 h. The infection rate of ASFV was determined by flow cytometry analysis (B), the genomic DNA level of the virus was determined by qPCR (C) and luciferase activity (D), and the virus titre in the culture supernatant was determined by TCID50 assay (E). The data were analysed statistically using one-way analysis of variance (ANOVA). The results are presented as the mean ± standard deviation of three independent measurements. (F) Co-IP and silver staining were used to detect the expression of endogenous p72 in PAMs and the…

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  Figure 2.. CD1d plays an important role for ASFV entry via binding to p72. (A–E) PAMs were transfected with scrambled si-Ctrl, and two specific RNAs targeting CD1d (si-CD1d-245 and si-CD1d-421) for 24 h, respectively. Then, the cells were infected with 1 MOI of rASFV-Gluc-GFP at 37°C for 24 h, the mRNA level of CD1d was examined by qPCR (A) and the protein level of CD1d was examined by Western blot (B). The genomic DNA level of ASFV in cells was determined by qPCR (C) and luciferase activity (D), and the virus titre in the culture supernatant was determined by TCID50 assay (E). (F–I) PAMs were incubated with anti-pCD1d antibody first at the indicated concentrations or IgG as a negative control for 1 h at 4°C, followed by infection with rASFV-Gluc-GFP at an MOI of 1. At 24 hpi, the infection rate of the ASFV was determined by flow cytometry analysis (F), the genomic DNA level of the ASFV in cells was determined by qPCR (G) and luciferase activity (H), and the infectious-virus titre in the culture supernatant was determined by TCID50 assay (I). (J–M) 1 MOI of rASFV-Gluc-GFP virions was preincubated with recombinant CD1d protein at the indicated concentrations or BSA as a negative control for…

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  Figure 3.. CD1d is involved in ASFV internalization. (A-C) MA104-WT and MA104-ΔCD1d cells were infected with rASFV-Gluc-GFP at an MOI of 1. At 24 hpi, the infection level of ASFV was determined by flow cytometry analysis (A), the genome level of the virus in cells was determined by qPCR (B) and luciferase activity (C). (D) MA104 and MA104-ΔCD1d cells were infected at 4°C with ASFV HLJ/2018 at an MOI of 2. After 1 h, cells were washed and collected for viral genome level (qPCR). Additional samples were then incubated at 37°C for 1 h, cells were washed and collected for viral genome level (qPCR). Incubation at 4°C with glycine (+Gly) represents the removement of the unbound virus from the cell surface. (E) PAMs were transfected with scrambled siRNA (si-Ctrl) and si-CD1d for 24 h, respectively. Then, the cells were infected with DiD-ASFV at 37°C. After 1 h, the cells were washed and collected for ASFV internalization by flow cytometry analysis. (F and G) PAMs were transfected with scrambled siRNA (si-Ctrl) and si-CD1d for 24 h, respectively. Then, cells were infected with DiD-ASFV at 4°C for 1 h, washed of free DiD-ASFV, and then transferred to 37°C to allow incorporation of DiD-ASFV up to 1…

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  Figure 4.. CD1d facilitates ASFV internalization via CME. (A–E) PAMs were transfected with scrambled siRNA control (si-Ctrl) and siRNAs targeting CHC (si-CHC) for 24 h, respectively. Then, the cells were infected with rASFV-Gluc-GFP at 37°C for 24 h, the mRNA level of CHC was examined by qPCR (A) and the protein level of CHC was examined by Western blot (B). The genomic level of ASFV in the cells was determined by qPCR (C) and luciferase activity (D), and the ASFV titre in the culture supernatant was determined by TCID50 assay (E). (F–I) PAMs (F) or MA104 (H) cells were incubated with different concentrations of CPZ for 30 min and then infected with ASFV. The genomic DNA copies of the ASFV in the cells was determined by qPCR. Cell viability of PAMs (G) or MA104 (I) cells was determined by WST-1 cell cytotoxicity assay kit (Beyotime). (J) MA104-WT and MA104-ΔCD1d cells were pretreated with CPZ (20 μM) for 30 min, then infected with ASFV HLJ/2018 at an MOI of 1 for 1 h at 4°C. Next, the cells were washed to remove any unbound ASFV and incubate at 37°C for 1 h. Viral genome level was detected by qPCR. DMSO was used as a control. The data were analysed statistically using one-way analysis of…

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  Figure 5.. EPS15 interacts with CD1d and is involved in ASFV internalization via CME. (A-B) The cell lysates from HEK293T cells overexpressing Flag-EPS15 and HA-CD1d (A) or HA-CD1dΔC (B) were subjected to immunoprecipitation with anti-Flag antibody. Immunoprecipitants and whole-cell lysates (Input) were immunoblotted with an anti-Flag and anti-HA antibodies, respectively. (C) The cell lysates from HEK293T cells overexpressing Flag-EPS15, HA-p72, and HA-CD1d were subjected to immunoprecipitation with anti-Flag antibody. Immunoprecipitants and whole-cell lysates (Input) were immunoblotted with an anti-Flag and anti-HA antibodies, respectively. (D) PAMs were infected with ASFV HLJ/2018 at an MOI of 5 or mock-infected for 48 h, and the cell lysates were subjected to immunoprecipitation with anti-p72 antibody. Immunoprecipitants and whole-cell lysates (Input) were immunoblotted with anti-p72, anti-CD1d, and anti-EPS15 antibodies, respectively. (E) PAMs were mock-infected or infected with ASFV for 1, 2, 4, 8, 12 and 24 h, and the mRNA levels of EPS15 in each time point in PAMs were analysed by qRT-PCR. (F-J) PAMs were transfected with si-Ctrl and si-EPS15s for 24 h, respectively. Then, the cells…

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  Figure 6.. The UIM domains of EPS15 are required for ASFV internalization. (A) The diagram shows functional regions of the porcine EPS15-WT and EPS15ΔUIM. UIM, ubiquitin-interacting domain. (B) The concise procedure for sample preparation and treatment. (C–E) MA104 cells were overexpressed with dsRed, dsRed-EPS15-WT and dsRed-EPS15-ΔUIM, respectively. The transfected cells were then infected with rASFV-Gluc-GFP (GFP-ASFV). At 24 hpi, the cells were analysed by flow cytometry (C), observed by fluorescence microscopy (D) (Bar = 10 μm), and manually calculated for viral infection rate (E). More than two hundred transfected cells were examined in each case. The data were analysed statistically using one-way analysis of variance (ANOVA). The results are presented as the mean ± standard deviation of three independent measurements. ****P < 0.0001; ***P < 0.001.

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  Figure 7.. Schematic illustration of CD1d in enhancing ASFV endocytosis as a host factor. ASFV virions enter host cells via host factor CD1d on the cell membrane. The p72 on the surface of the ASFV virion binds with CD1d on the cell membrane. The signal is then transmitted to EPS15 in the cytoplasm to form the p72-CD1d-EPS15 axis, where CD1d recruits EPS15 through the interaction between CD1d and EPS15 intracellular domains. The process promotes the formation of EPS15-AP-2-clathrin complexes and aggregation, resulting in ASFV virions endocytosis during ASFV infection.