PCV2 Uptake by Porcine Monocytes Is Strain-Dependent and Is Associated with Amino Acid Characteristics on the Capsid Surface

Figures

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  FIG 1. PCV2 uptake by peripheral blood monocytes of purebred Piétrain pigs in a strain-dependent manner. (A) Visualization of the PCV2 uptake by monocytes. PCV2 capsid proteins were stained in green, and monocytes were stained in red. Representative confocal images are given for each strain and each time point. (B to F) Analysis of the ability of blood monocytes to take up viral particles of different PCV2 strains. All data are the mean values of 3 replicates (3 pigs; each replicate is the mean of 10 randomly selected fields). (B) The percentage of PCV2 positive cells at each time point was calculated in 10 randomly selected fields. (C) The mean fluorescing area of capsid proteins in a PCV2 positive CD172a+ cell at each time point was calculated in the form of digital pixels with MATLAB. (D) The Area Under the Curve (AUC) of each strain was calculated. (E) Quantification of PCV2 particles that are attached to monocytes. The mean fluorescing area of viral capsids outside the cell was calculated. (F) Quantification of PCV2 particles that internalized into monocytes. The mean fluorescing area of viral capsids inside the cell was calculated. PCV2 particles were scored as internalized once…

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  FIG 2. Expression of heparan sulfate (HS), chondroitin sulfate (CS), and decorin (linked with dermatan sulfate [DS] chains) on PK15 cells and peripheral blood monocytes of purebred Piétrain and Landrace pigs. Data were obtained from 3 different wells for PK-15 cells, and 3 pigs of each breed for monocytes. (A) Confocal images showing PK15 cells and peripheral blood monocytes stained with specific antibodies against HS, CS, and decorin. (B) Quantification of the percentage of PK-15 cells and monocytes expressing HS, CS, decorin, and the mean fluorescing area (pixels) of GAG per GAG+ cell. Data represent the means ± SD of 3 experiments. Differences between PK-15 cells and monocytes were revealed by two-way ANOVA with Dunnett’s post-test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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  FIG 3. Colocalization of GAG (HS, CS-A/C, decorin [DS]) and PCV2 particles. Blood monocytes from 3 Piétrain pigs were incubated with PCV2 particles at 4°C for 1 h, after which the virus was removed, and cells were fixed with 4% PF. Double immunofluorescence staining to detect GAG and PCV2 capsid proteins was performed. GAG was stained in green, and the PCV2 virions were stained in red. Cell nuclei were co-stained in blue. Visualization of colocalization was performed by confocal microscopy. White arrows indicate the colocalization between GAG and PCV2 particles in merged images. All data are the mean values of 3 replicates (3 pigs; each replicate is the mean of 10 randomly selected fields). (A) Representative pictures of cells with 1121 and DE222-13 PCV2 particles colocalized with GAG. (B) The percentage of PCV2-antigen-positive cells was determined. (C) Fluorescing area of attached PCV2 particles per cell was quantified by ImageJ in pixels. (D) The percentage of cells showing colocalization between PCV2 particles and GAG was determined. (E) For each PCV2-GAG colocalized cell, the proportion of PCV2 positive pixels that are also GAG positive was assessed based on individual fluorescing…

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  FIG 4. Effects of GAGs on virus attachment (A, B, and C) and internalization (D, E, and F) on/in blood monocytes from 3 Piétrain pigs. Equal amounts of PCV2 particles were mixed with individual GAGs or GAG combinations at 37°C for 90 min before being added to monocytes for an incubation period of 1 h at either 4°C or 37°C. For confocal microscopy, 10 random fields of each slide/each pig were selected for quantification. (A and D) Visualization of PCV2 particles attached to the monocyte surface (A)/internalized into monocytes (D) was conducted by LSCM. Bar = 10 μm. (B and E) The percentage of monocytes with PCV2 sticking on the cell surface (B)/internalization into the cell (E) was counted. (C and F) The fluorescing area of attached (C)/internalized (F) viral particles per cell was quantified with MATLAB. Data represent the means ± SD of 3 independent experiments (3 pigs). For each strain, every bar was compared with its Mock, and the significance of the differences was calculated by the Student's t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

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  FIG 5. Effects of enzymatic removal of CS-A/B/C and/or HS on PCV2 attachment (A, B, and C) onto the surface of blood monocytes, and on the PCV2 internalization (D, E, and F) into these cells. Cells were isolated from 3 purebred Piétrain pigs, and pretreated with either chondroitinase ABC or heparinase I&III, or the combination of the 2 enzymes at 37°C for a 1-h treatment before virus incubation at either 4°C or 37°C for 1 h. For confocal microscopy, 10 random fields of each slide/each pig were selected for quantification. (A and D) Visualization of PCV2 attachment onto (A)/internalization into (D) monocytes. Bar = 10 μm. (B and E) The percentage of cells showing PCV2 attachment (B)/internalization (E) was determined. (C and F) The fluorescing area of attached (C)/internalized (F) virus particles per cell was quantified with MATLAB. Data represent the means ± SD of 3 experiments (3 pigs). For each strain, every bar was compared with its Mock, and the significance of the differences was estimated by Student's t test. *, P < 0.05; **, P < 0.01.

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  FIG 6. Three dimensional mapping of the monomer, trimer, and pentamer of the PCV2 capsid protein of the PCV2 strains PCV2a (Stoon-1010), PCV2a (1121), PCV2b (1147), PCV2d-1 (09V448), PCV2d-2 (DE222-13), and PCV2d-2 (19V245). Charged residues were labeled in 2 major colors: positively charged residues (arginine [R], lysine [K], histidine [H])-red; negatively charged residues (aspartic acid [D], glutamic acid [E]-blue. The upper row presents the outer surface of the PCV2 capsid monomer. The middle rows present the outer surface of the PCV2 capsid trimer and the magnification of 1 of the positively charged bands of the three-wings-windmill-like pattern. Two configurations were found based on the specific positively charged bands in the middle of these models (circled in green). Pattern 1 shows an incompact and disconnected pattern, while pattern 2 shows a connected and regular three-wing-windmill pattern. The lower rows present the outer surface of the PCV2 capsid pentamer, and the magnification of 1 of the positively charged patches of the pentagon pattern. The amino acids composing the pentagon were circled in green. The 3D structures of monomeric capsid were predicted using the I-TASSER…

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  FIG 7. Expression of phosphacan on HepG2 cells (positive control), PK-15 cells, and peripheral blood monocytes of Piétrain and Landrace pigs, and colocalization of CS and phosphacan. Monocytes were fixed with 4% PF at RT for 10 min, followed by double immunofluorescence staining with specific primary antibodies and conjugated secondary antibodies. (A) Confocal images showing HepG2 cells, PK-15 cells, and monocytes stained with a specific antibody against phosphacan. (B) Quantification of the percentage of cells expressing phosphacan, and the mean fluorescing area (pixels) of phosphacan per phosphacan+ cell. (C) Representative images showing colocalization of CS and phosphacan on monocytes. White arrows indicate the colocalization between CS and phosphacan in merged images. (D) Quantification of the percentage of cells showing colocalization of CS and phosphacan. Data represent the means ± SD of 3 experiments. Differences between Piétrain and Landrace were revealed by the Student's t test. *, P < 0.05; ***, P < 0.001.

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  FIG 8. Colocalization of PCV2 and phosphacan. Monocytes were incubated with PCV2 particles at 4°C for 1 h, after which the virus was removed and cells were fixed with 4% PF. Phosphacan was stained in green, and PCV2 particles were stained in red. Cell nuclei were co-stained in blue. (A) Representative pictures of cells with 1121 and DE222-13 particles showing colocalization with phosphacan. White arrows indicate the colocalization between PCV2 virions, and phosphacan in merged images. (B) The percentage of cells showing colocalization between PCV2 particles and phosphacan was determined. (C) For each PCV2-phosphacan colocalized cell, the proportion of PCV2 positive pixels that are also phosphacan positive was quantified based on their individual fluorescing pixels per cell. Data represent the means ± SD of 3 experiments. Differences between 1121 and DE222-13 were revealed by the Student's t test. **, P < 0.01.

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  FIG 9. Hypothetical model of PCV2 uptake mediated by phosphacan and other potential molecules. (A) To begin the process of virus uptake, the PCV2 virion electrostatically attaches to the GAG chains of phosphacan, a cell surface proteoglycan. Any 3 chains on phosphacan may interact with the three-wings-windmill-shaped positively charged bands (colored in red in capsid trimer) on the surface of the PCV2 capsids, allowing viral particles to accumulate on the monocyte surface. Given that it has fewer GAG chains and endocytic motifs, CSPG2/NG2 will have poorer binding and internalization efficiency. Extracellular decorin and biglycan may support viral enrichment on the cell surface, and viral delivery to other binding and internalization mediators. Afterwards, the interaction of the viral particle with phosphacan activates the signaling pathway of clathrin-mediated endocytosis, and induces the binding of an adaptor protein complex 2 (AP2) to the phosphacan cytoplasmic tail. APs bind to clathrin, and allow clathrin to involve in forming clathrin-coated vesicles (CCVs) under the facilitation of dynamin. The vesicle then delivers its viral content to endosomes. Then, the endosome with the virus…