Early Porcine Sapovirus Infection Disrupts Tight Junctions and Uses Occludin as a Coreceptor

Figures

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  FIG 1. Early PSaV infection dissociates tight junctions. (A) TER was determined in confluent LLC-PK monolayers grown on transwell inserts treated with medium only (mock-treated group) or with 200 μM GCDCA bile acid and infected or not with PSaV strain Cowden (MOI = 50) or PSaV VLPs (4 μg/ml) at the indicated time points. The data are presented as means and standard errors of the mean. (B) Paracellular flux of 4-kDa FITC-dextran and 70-kDa FITC-dextran was measured in the apical-to-basolateral direction of the confluent LLC-PK cells grown on transwell inserts treated or not with 200 μM GCDCA bile acid and the calcium ion chelating agent EGTA (1.8 mM) and infected or not with PSaV strain Cowden (MOI = 50) in the presence or absence of 200 μM GCDCA or PSaV VLP (4 μg/ml). The amount of tracer diffusion was normalized to that of mock-treated, mock-inoculated control LLC-PK cells. The error bars indicate standard errors of the mean. (C) LLC-PK cells grown on the transwell inserts were mock treated (medium only) as a negative control, treated with 1.8 mM EGTA and incubated for 10 min, or infected with the PSaV strain Cowden (MOI = 50) in the presence of 200 μM GCDCA or PSaV VLPs (4 μg/ml) and…

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  FIG 2. Occludin is internalized in response to PSaV infection. (A) LLC-PK monolayers infected or not with PSaV strain Cowden (MOI = 50) in the presence or absence of 200 μM GCDCA bile acid were incubated for 30 min or 60 min; fixed; and stained for occludin, claudin-1, JAM-1, and ZO-1 using each corresponding antibody. Scale bars, 50 μm. (B) Quantification of the internalization of occludin, claudin-1, JAM-1, and ZO-1 under the conditions for panel A was determined by counting cells containing each TJ protein in the cytoplasm and expressed as a percentage of the total cell numbers. The error bars indicate standard errors of the mean. (C) Confluent LLC-PK monolayers infected or not with PSaV (MOI = 50) in the presence or absence of 200 μM GCDCA bile acid were fixed and stained for occludin and ZO-1 at 30 min p.i. The cells were observed by x-sectioning (left) or z-sectioning (right) under a confocal microscope. Scale bars, 10 μm. All experiments were performed in triplicate, and representative images are shown in panels A and C.

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  FIG 3. Mean numbers of viable virions were determined by plaque and quantitative real-time PCR assays before and afterAF594 labeling. (A and B) Purified PSaV strain Cowden (MOI = 50) was either labeled with AF594 or left unlabeled, and the virus titer was checked by plaque (A) and quantitative real-time PCR (B) assays as described in Materials and Methods. The error bars indicate standard errors of the mean.

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  FIG 4. PSaV colocalizes and coprecipitates with occludin. (A) LLC-PK cells were inoculated with Alexa Fluor 594-labeled PSaV strain Cowden (4 × 102 particles/cells) for 1 h on ice and shifted to 37°C for 30 min. The cells were fixed and incubated with antibodies specific for occludin (OCC), claudin-1 (CLD-1), JAM-1, or ZO-1. After washing twice with PBS-FBS, the cells were incubated with FITC-conjugated secondary antibodies at 20°C for 1 h. The cells were mounted with SlowFade Gold antifade reagent containing 1× DAPI solution for staining of nuclei. The slides were analyzed immediately by x-sectioning under a confocal microscope. One representative set of results is shown. The scale bars represent 20 μm. (B) The lysates of PSaV-infected LLC-PK cells were immunoprecipitated with antibodies specific for OCC, JAM-1, ZO-1, or CLD-1. As a mock control, immunoglobulin G was used for immunoprecipitation (IP) of the virus-infected cell lysates. The coimmunoprecipitated proteins were analyzed by Western blotting to detect OCC, JAM-1, ZO-1, or CLD-1 using the relevant antibody. To confirm the presence of each target protein in the cell lysates (input), the lysates of PSaV-infected LLC-PK cells…

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  FIG 5. Occludin functions as a coreceptor for PSaV. LLC-PK cells were incubated with antibody against OCC, CLD-1, or a control IgG for 1 h at 4°C before (A, B, E, and F) or after (C, D, G, and H) attachment of PSaV strain Cowden (MOI = 50). After washing the cells with cold PBS, infection was allowed to proceed, and the cells were harvested at the indicated time points. Quantitative real-time PCR (A, C, E, and G) and TCID50 analyses (B, D, F, and H) were used to evaluate PSaV genome copy numbers and titers, respectively. All experiments were performed in triplicate. The data are presented as means and standard errors of the mean. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

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  FIG 6. siRNAs against occludin and claudin-1 block PSaV entry. (A) LLC-PK cells at 70 to 80% confluence were transfected with 100 pmol of scrambled siRNA or 4 individual pooled siRNAs against OCC, CLD-1, JAM-1, or ZO-1. Cells were harvested 24 or 48 hpt, and Western blot analyses were performed to determine the downregulation of each target gene. GAPDH was used as a loading control. The mean fold change (MFC) was calculated using GelQuant.NET software (BiochemLab Solutions) by comparing the relative density of siRNA-treated samples to that of siScram-treated samples. (B) Monolayers of LLC-PK cells in 8-well chamber slides were transfected with siScram or siRNAs against OCC, CLD-1, JAM-1, or ZO-1 and stained with the relevant antibodies. The cells were mounted with SlowFade Gold antifade reagent containing 1× DAPI solution for staining of nuclei. The slides were analyzed immediately by x-sectioning under a confocal microscope. Scale bars, 20 μm. (C) Confluent LLC-PK cells in 8-well chamber slides were infected with Alexa Fluor 594-labeled PSaV strain Cowden (400 particles per cell) 48 h after transfection with scrambled, occludin (siOCC), claudin-1 (siCLD-1), JAM-1 (siJAM-1), or ZO-1…

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  FIG 7. Determining off-target effects of siRNA against occludin. (A) Nucleotide sequences of wild-type (GFP-WT-OCC) and mutant (GFP-M1-OCC and GFP-M2-OCC) occludins and each siRNA. Using the plasmid encoding wild-type GFP-tagged occludin at the 5′ end (GFP-WT-OCC) and two mutant plasmids (GFP-M1-OCC and GFP-M2-OCC) containing four silent-mutation sites, each siRNA was generated by site-directed mutagenesis using primer pairs containing mutation sites as described in Materials and Methods. The red letters indicate the locations of mutation sites, and magenta letters indicate the unchanged siRNA sequence. (B and C) LLC-PK cells grown at 70 to 80% confluence on 8-well chamber slides were transfected with GFP-WT-OCC or GFP-M1-OCC and GFP-M2-OCC mutant plasmids. At 4 hpt, individually with each plasmid, the cells were then transfected with 4 individual pooled siRNAs against occludin, and knockdown effects by occludin siRNAs were examined by confocal microscopy (B) and Western blot analyses (C) using antibody specific for occludin as described in Materials and Methods. GAPDH was used as a loading control in panel C. The scale bars in panel B represent 20 μm.

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  FIG 8. siRNAs against occludin and claudin-1 block PSaV infection. LLC-PK cells at 70 to 80% confluence grown in 6-well plates were infected with PSaV strain Cowden (MOI = 50) 48 h after transfection with 100 pmol siScram, siOCC, siCLD-1, siJAM-1, or siZO-1 and incubated in the presence of 200 μM GCDCA for the indicated times. (A) The PSaV genome copy numbers at each harvest time point were determined by quantitative real-time PCR, and the inhibitory effect of each siRNA on PSaV genome replication was compared with that of scrambled-siRNA-transfected, PSaV-infected cells. (B) LLC-PK cells grown in 96-well plates were inoculated with 200 μl of 10-fold serial dilutions of virus-containing supernatant and incubated at 37°C for 6 days. Virus titers were determined by the Reed-Muench method and expressed as TCID50 per milliliter. The inhibitory effect of each siRNA on the PSaV titer was compared with that of scrambled-siRNA-transfected, PSaV-infected cells. (C) The cell lysates harvested at the indicated time points were analyzed by Western blotting using antibody specific for PSaV VPg protein. GAPDH was used as a loading control. The MFC was calculated using GelQuant.NET software by…

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  FIG 9. Synergistic effect of combined siRNA against occludin and claudin-1. (A) LLC-PK cells grown in 6-well plates at 70 to 80% confluence were transfected with combined siRNAs against occludin and claudin-1 or scrambled siRNA. At 48 hpt, cells were harvested, and knockdown of their target proteins was assessed by Western blot analysis. GAPDH was used as a loading control. The MFC was calculated using GelQuant.NET software by comparing the relative density of siRNA-treated samples to that of siScram-treated samples. (B to D) LLC-PK cells were transduced with combined siRNAs against occludin and claudin-1 for 48 h. Next, the cells were infected with PSaV strain Cowden (MOI = 50) in the presence of 200 μM GCDCA for the indicated times. (B) The PSaV genome copy numbers at the indicated time points were determined by quantitative real-time PCR, and the inhibitory effects of combined siRNAs against occludin and claudin-1 were compared with those of scrambled-siRNA-transfected and PSaV-infected cells. (C) LLC-PK cells grown on 96-well plates were infected with 200 μl of 10-fold serial dilutions of virus-containing supernatant and incubated at 37°C for 6 days. Virus titers were determined by…

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  FIG 10. PSaV infection in nonpermissive cells expressing occludin. (A) CHO cells were transiently transduced with 1 μg of pCDNA3.1 or pmRFP1-occludin (pmRFP1-OCC), and expression of occludin was checked by confocal microscopy using antibody specific for occludin. (B) CHO cells grown in 8-well chamber slides were transfected with pCDNA3.1 or pmRFP1-OCC plasmid and then incubated for 24 h. The cells were then infected with PSaV (MOI = 50) and incubated for the indicated times. The cells were washed, fixed with 4% paraformaldehyde, permeabilized with 0.01% Triton X-100, and then stained with antibody specific for PSaV VPg. The cells were mounted with SlowFade Gold antifade reagent containing 1× DAPI solution for staining of nuclei. The slides were analyzed immediately by x-sectioning under a confocal microscope. (C) CHO cells grown in 6-well plates at 70 to 80% confluence were transfected with 1 μg pmRFP1-OCC. At 24 h posttransfection, the cells were infected with PSaV strain Cowden (MOI = 50) and incubated for the indicated times. LLC-PK monolayers grown in 6-well plates were inoculated with PSaV strain Cowden (MOI = 50) and incubated for 24 h. The cell lysates harvested at the specified…

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  FIG 11. Coentry of occludin and PSaV depends on Rab5. (A) Confluent LLC-PK cells grown in 8-well chamber slides were inoculated with Alexa Fluor 594-labeled PSaV (MOI = 50), incubated for 1 h at 4°C, and then incubated for another 30 min at 37°C. Confocal microscopy was performed to detect the colocalization of Alexa Fluor 594-labeled PSaV and the early endosome marker EEA1. Scale bars, 50 μm. (B) LLC-PK cells at 70 to 80% confluence were infected with Alexa 594-labeled PSaV (MOI = 50) 48 h after transfection with scrambled siRNA or siRNAs against Rab5 and then incubated at 37°C for 30 min. The knockdown effects on Rab5 protein by scrambled siRNA or siRNAs against Rab5 was evaluated by Western blot analysis. GAPDH was used as a loading control. The MFC was calculated using GelQuant.NET software by comparing the relative density of siRNA-treated samples to that of scrambled-siRNA-transfected samples. Scale bars, 50 μm. (C) LLC-PK cells transfected with either wild-type EGFP-Rab5 or a dominant-negative mutant (S34N) plasmid were infected with Alexa Fluor 594-labeled PSaV (MOI = 50) and stained for occludin (white) at 30 min postinoculation. The cells were mounted with SlowFade Gold…

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  FIG 12. Coentry of occludin and PSaV depends on Rab7. (A) Confluent LLC-PK cells grown in 8-well chamber slides were inoculated with Alexa Fluor 594-labeled PSaV (MOI = 50), incubated for 1 h at 4°C, and then incubated for another 60 min at 37°C. Colocalization of Alexa 594-labeled PSaV and the late endosome marker LAMP2 was evaluated by confocal microscopy. Scale bars, 50 μm. (B) LLC-PK cells at 70 to 80% confluence were infected with Alexa Fluor 594-labeled PSaV (MOI = 50) 48 h after transfection with scrambled siRNA or siRNA against Rab7 and then incubated at 37°C for 30 min. The knockdown effects on Rab7 protein by scrambled siRNA or siRNAs against Rab7 were evaluated by Western blot analysis. GAPDH was used as a loading control. The MFC was calculated using GelQuant.NET software by comparing the relative density of siRNA-treated samples to that of scrambled-siRNA-transfected samples. Scale bars, 50 μm. (C) LLC-PK cells transfected with either wild-type-form EGFP-Rab7 or a dominant-negative mutant (T22N) were infected with Alexa Fluor 594-labeled PSaV particles (MOI = 50) and stained for occludin (white) at 60 min postinoculation. The cells were mounted with SlowFade Gold antifade…