ADAM17 is an essential attachment factor for classical swine fever virus

Figures

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  Fig 1. Identification of ADAM17 by pull-down with sE2-Fc. (A) Coomassie blue staining of sE2-Fc with or without DTT treatment. (B) Binding of sE2-Fc to PK15 cells, BHK-21 cells, PK15 cells in the presence of anti-integrin antibody JSB5 or anti-CSFV antibody V3 by flow cytometry analysis. Mean fluorescence intensity (MFI) values were determined using FlowJo software and normalized to MFI of PK15. (C) PK15 cells were pretreated with sE2-Fc (left) or BSA (right) followed by pseudoparticle infection. Error bars indicate standard deviation (SD) of mean (n = 3). The data represent three independent experiments. (D) Pull-down of sE2-Fc binding proteins from PK15 cell lysates solubilized by various detergents. The membrane proteins were labeled with sulfo-NHS-LC-biotin, and blotted with HRP-conjugated streptavidin.

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  Fig 2. ADAM17 is required for CSFV entry into porcine cells. (A) Binding of sE2-Fc to PK15, PK15 ADAM10 knockout (ADAM10-KO), PK15 ADAM17 knockout (ADAM17-KO), or pig ADAM17 trans-complemented ADAM17-KO cells (ADAM17-KO+pADAM17) by flow cytometry. (B) Cell lines indicated above were infected with lentiviruses encoding a GFP reporter and pseudotyped with envelope proteins from CSFV SXCDK (CSFVpp-SXCDK) or Shimen (CSFVpp-Shimen) strains. The infection titer was measured as focus-forming units per ml (FFU/ml). Pseudoparticles enveloped with VSV G protein (VSV-Gpp) served as the positive control, and that bearing no envelope protein (Non-envelope) was the negative control. (C) Indicated cells were infected with CSFVcc C strain or recombinant VSV encoding GFP reporter (rVSV-eGFP-G). Viral infectivity was expressed as E2 or GFP-positive foci. (D) Relative CSFVpp-Shimen infection of poorly permissive porcine cell lines with or without ADAM17 overexpression. (E-F) Porcine primary embryonic fibroblasts were infected with CSFVcc 60 h after transfection with irrelevant (siCon.), or ADAM17-specific siRNAs (siADAM17-1 and siADAM17-3). ADAM17 mRNA levels (E) and CSFV RNA in the supernatants (F) were…

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  Fig 3. ADAM17 expression correlates with CSFV susceptibility. (A) PK15, ST, 3D4/21 and IPEC-J2cells were infected with the indicated pseudoparticles. (B) The expression levels of ADAM17 and TIMP-3 were measured by qRT-PCR. The mRNA level in PK15 cells was set as 100%. (C) Silencing of TIMP-3 in ST cells with two oligos (siTIMP3-2 and siTIMP3-3). TIMP-3 mRNA levels were measured by qRT-PCR and expressed as a percentage of control oligo (siCon.). (D) CSFVpp-SXCDK and VSV-Gpp infection of siRNA-treated ST cells normalized to siCon.-treated cells. Error bars indicate standard deviation (SD) of the mean (n = 3). Significance was calculated using a multiple t test. The data represent three independent experiments.

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  Fig 4. E2 binds the metalloproteinase domain of ADAM17. (A) PK15 cells were treated with indicated concentrations of the ion chelator: 1, 10-phenanthroline, and sE2-Fc binding was tested by flow cytometry analysis. ADAM17-KO cells transduced with hADAM17 WT or H405D mutant were processed for sE2-Fc binding (B) and pseudovirus infection (C). The lower limit of detection was 10 FFU/ml (dashed line). Asterisks indicate samples below the limit. (D) PK15 cells were treated with indicated concentrations of aderbasib, and sE2-Fc binding was determined by flow cytometry analysis. Binding between soluble ADAM17 metalloproteinase domain (sMP) and sE2 was characterized by surface plasmon resonance (SPR) with (F) or without (E) zinc ions in the running buffer. Experimental curves (red lines) were fit using a 1:1 binding model (black lines). The equilibrium dissociation constant (KD) were calculated by BIAcore T100 Evaluation software. CSFVpp (G) and VSV-Gpp (H) infection of PK15 cells pretreated with sMP or BSA. Error bars indicate SD of the mean (n = 3). The above data represent three independent experiments.

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  Fig 5. Key region in ADAM17 responsible for CSFV entry. (A) Schematic diagram of pADAM17, zfADAM17 and construction of the chimera ADAM17s. Chimera ADAM17s were constructed by replacing the indicated fragments of zfADAM17 with those of pADAM17. SP, signal peptide; PRO, pro-domain; MP, metalloproteinase domain (N223-V477); DD, disintegrin domain; CD, catalytic domain; TM, transmembrane domain; ICD, intracellular domain. (B) Relative binding of sE2-Fc with PK15 (WT), ADAM17-KO and ADAM17-KO stably transduced with pADAM17 (+pADAM17), zfADAM17 (+zfADAM17) or various ADAM17 chimeras by flow cytometry analysis. (C) Above cell lines were infected with indicated pseudoparticles. The lower limit of detection was 10 FFU/ml (dashed line). Asterisks indicate samples below the limit. (D) Western blotting of chimera ADAM17s in the cell lysates using anti-Flag antibody, with anti-GAPDH antibody as the control. Error bars in (B) and (C) indicate SD of the mean (n = 3). The data represent three independent experiments.