Porcine Sapovirus-Induced Tight Junction Dissociation via Activation of the RhoA/ROCK/MLC Signaling Pathway

Figures

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  FIG 1. Early PSaV infection induces phosphorylation of MLC in a RhoA/ROCK-dependent manner in polarized LLC-PK cells. (A and B) Confluent LLC-PK cells were mock infected or infected with the PSaV Cowden strain (MOI of 1) in the presence of 200 μM GCDCA, and cells were harvested at the indicated time points. (A) Cell lysates were subjected to Western blotting to determine the expression levels of pMLC and MLC2 using the relevant antibodies or (B) were subjected to a RhoA activation assay to measure RhoA activation with an anti-RhoA antibody. The expression level of ROCK in the cell lysate was analyzed by Western blotting using an antibody against ROCK. (C and D) Confluent LLC-PK cells were mock treated or pretreated with RhoA inhibitor (CT04) (C) or ROCK inhibitor (Y27632) (D) at the indicated doses for 1 h at 37°C. These cells were then mock infected or infected with PSaV Cowden strain (MOI of 1) in the presence of 200 μM GCDCA. Cell lysates were harvested at 30 mpi, and the expression levels of ROCK and/or pMLC were evaluated by Western blotting. GAPDH was used as a loading control. Representative images of different gels from each group are presented. Data are presented as means ±…

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  FIG 2. Early RVA infection induces phosphorylation of MLC in a RhoA/ROCK-dependent manner in polarized MDCK cells. (A and B) Confluent MDCK cells were mock infected or infected with bovine RVA NCDV strain (MOI of 10), and cells were harvested at the indicated time points. (A) Cell lysates were subjected to Western blotting to determine the expression levels of pMLC and MLC2 using the relevant antibodies or (B) were subjected to a RhoA activation assay to measure RhoA activation with an anti-RhoA antibody. The expression level of ROCK in the cell lysate was analyzed via Western blotting by using an antibody against ROCK. (C and D) Confluent MDCK cells were mock treated or pretreated with RhoA inhibitor (CT04) (C) or ROCK inhibitor (Y27632) (D) at the indicated doses for 1 h at 37°C. These cells were then mock infected or infected with bovine RVA NCDV strain (MOI of 10). Cell lysates were harvested 1 h postinfection, and the expression levels of ROCK and/or pMLC were evaluated via Western blotting. GAPDH was used as a loading control. Representative images of different gels from each group are presented. Data are presented as means ± standard errors of the mean from three independent…

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  FIG 3. Determination of the cytotoxicity of chemicals by the MTT assay. (A to E) LLC-PK cells grown in 96-well plates were incubated with various concentrations of the indicated chemicals in triplicate for 24 h at 37°C. Afterward, the chemical-containing media were thoroughly removed and replaced with 200 μl of MTT solution for 4 h at 37°C. Each well was incubated with 150 μl of DMSO for 10 min at 20°C. Cell viability was measured as OD570 using an ELISA reader.

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  FIG 4. PSaV infection activates the RhoA/ROCK/MLC signaling pathway in the absence of GCDCA. (A and B) PSaV Cowden strain (MOI of 1), in the absence of GCDCA, was inoculated into confluent LLC-PK cells, and cells were harvested at the indicated time points. (A) Cell lysates were subjected to Western blotting to determine the expression levels of pMLC and MLC2 using the relevant antibodies or (B) were subjected to a RhoA activation assay to measure RhoA activation with an anti-RhoA antibody. The expression level of ROCK in the cell lysate was analyzed by Western blotting using an antibody against ROCK. (C and D) Confluent LLC-PK cells were pretreated with or without RhoA inhibitor (CT04) (C) or ROCK inhibitor (Y27632) (D) at the indicated doses for 1 h at 37°C and then mock infected or infected with PSaV Cowden strain (MOI of 1) in the absence of GCDCA. Cell lysates were harvested at 30 mpi, and the expression levels of ROCK and/or pMLC were evaluated via Western blotting. GAPDH was used as a loading control. Representative images of different gels from each group are presented. Data are presented as means ± standard errors from three independent experiments. The band intensities of…

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  FIG 5. MYPT subunit is not activated in LLC-PK cells during the early stage of PSaV infection. (A and B) Confluent LLC-PK cells were mock infected or infected with PSaV Cowden strain (MOI of 1) in the presence (A) or absence (B) of GCDCA. Cells were then harvested at the indicated time points. The cell lysates were subjected to Western blotting to determine the expression levels of pMYPT and MYPT using the relevant antibodies. GAPDH was used as a loading control. Representative images of different gels from each group are presented. Data are presented as means ± standard errors from three independent experiments. The band intensities of pMYPT1, relative to GAPDH, were determined using densitometric analysis across three independent experiments and statistically analyzed and are shown as graphs below each Western blot. Differences were evaluated using a one-way ANOVA. (C to E) Lysates of LLC-PK cells either mock infected or infected with PSaV Cowden strain (MOI of 1) in the presence (C) or absence (D) of GCDCA at the indicated time points and lysates of MDCK cells either mock infected or infected with bovine RVA NCDV strain (MOI of 10) (E) were immunoprecipitated using antibodies…

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  FIG 6. PSaV-induced activation of pMLC is independent of the PKC/MLCK signaling pathway. (A and B) Confluent LLC-PK cells were either mock infected or infected with PSaV Cowden strain (MOI of 1) in the presence (A) or absence (B) of GCDCA for the indicated time points. Cell lysates were subjected to Western blotting to detect pPKC and MLCK using the relevant antibodies. GAPDH was used as a loading control. (C and D) Confluent LLC-PK cells were either mock treated or pretreated with the PKC inhibitor Gö 6983 or the MLCK inhibitor ML7 for 1 h at 37°C and then infected with PSaV Cowden strain (MOI of 1) in the presence (C) or absence (D) of GCDCA. Cell lysates were harvested after 30 min, and the expression level of pMLC was evaluated via Western blotting. GAPDH was used as a loading control. Representative images of different gels from each group are presented. Data are presented as means ± standard errors from three independent experiments. The band intensities of pPKC and MLCK, relative to GAPDH, were determined using densitometric analysis across three independent experiments and statistically analyzed and are shown as graphs below each Western blot. Differences were evaluated using a…

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  FIG 7. Rotavirus-induced activation of pMLC is independent of the PKC/MLCK signaling pathway. (A) Confluent MDCK cells were either mock infected or infected with bovine RVA NCDV strain (MOI of 10) for the indicated time points. Cell lysates were subjected to Western blotting to detect pPKC and MLCK by using the relevant antibodies. GAPDH was used as a loading control. (B) Confluent MDCK cells were either mock treated or pretreated with the PKC inhibitor Gö 6983 or the MLCK inhibitor ML7 for 1 h at 37°C and then infected with bovine RVA NCDV strain (MOI of 10). Cell lysates were harvested after 1 h, and the expression level of pMLC was evaluated via Western blotting. GAPDH was used as a loading control. Representative images of different gels from three independent experiments are presented. Data are presented as means ± standard errors from three independent experiments. The band intensities of pPKC and MLCK, relative to GAPDH, were determined using densitometric analysis across three independent experiments and statistically analyzed and are shown as graphs below each Western blot. Differences were evaluated using a one-way ANOVA.

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  FIG 8. Inhibition of the RhoA/ROCK/MLC signaling pathway affects PSaV infectivity and viral protein expression. LLC-PK cells were mock treated or pretreated with noncytotoxic concentrations of RhoA inhibitor CT04 (2 μg), ROCK inhibitor Y27632 (20 μM), or MLC inhibitor blebbistatin (10 μM) for 1 h at 37°C and then infected with PSaV Cowden strain (MOI of 1) in the presence of 200 μM GCDCA for 36 h. (A) Genome copy number was determined via RT-qPCR. (B) Virus titers were determined using TCID50 using cell lysates produced by three cycles of freezing and thawing. (C) VPg was detected via Western blotting. PSaV genome copy numbers, viral titers, and VPg expression levels were described in Materials and Methods. GAPDH was used as a loading control. The band intensity of VPg, relative to GAPDH, was determined using densitometric analysis across three independent experiments and statistically analyzed and is shown as a graph below the Western blot. All experiments, including RT-qPCR and TCID50 assays, were also performed in triplicate. Representative images of different gels from each group are presented. Data are presented as means ± standard errors from three independent experiments.…

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  FIG 9. Inhibition of the RhoA/ROCK/MLC pathway restores the TER of the TJ in PSaV-infected polarized epithelial cells. (A and B) Confluent LLC-PK cells grown on Transwell filters were pretreated with or without RhoA inhibitor CT04 (2 μg), ROCK inhibitor Y27632 (20 μM), or MLC inhibitor blebbistatin (10 μM) for 1 h at 37°C and then mock inoculated or inoculated with PSaV Cowden strain (MOI of 50) in the presence (A) or absence (B) of GCDCA, followed by determination of TER at the indicated time points. Net TER was calculated by subtracting the background (membrane filter without cells) and multiplying the resistance (ohms) by the area (0.33 cm2) of the filter. All experiments were performed in triplicate. Data are presented as means ± standard errors from three independent experiments.

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  FIG 10. Distribution of occludin is altered by the PSaV-induced RhoA/ROCK/MLC signaling pathway. (A to D) Confluent LLC-PK cells were untreated or treated with RhoA inhibitor CT04 (2 μg), ROCK inhibitor Y27632 (20 μM), or MLC inhibitor blebbistatin (10 μM) for 1 h at 37°C and then mock inoculated or inoculated with PSaV Cowden strain (MOI of 50) in the presence (A and B) or absence (C and D) of GCDCA for 30 min. Cells were then fixed, permeabilized, and prepared for confocal microscopy using rabbit antioccludin, anticlaudin, anti-JAM-A, and anti-ZO-1 antibodies and relevant secondary antibodies. All experiments were performed in triplicate and representative images are shown. The scale bars correspond to 10 μm. (B and D) The internalized TJ molecules, shown as a percentage of intracellular fluorescence/total fluorescence, are presented as means ± standard errors from three independent experiments. Differences were evaluated using a one-way ANOVA. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

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  FIG 11. Distribution of the TJ proteins is altered by the rotavirus-induced RhoA/ROCK/MLC signaling pathway. (A and B) Confluent MDCK cells were untreated or treated with RhoA inhibitor CT04 (2 μg), ROCK inhibitor Y27632 (20 μM), or MLC inhibitor blebbistatin (10 μM) for 1 h at 37°C and then mock inoculated or inoculated with bovine RVA NCDV strain (MOI of 10) for 1 h. Cells were then fixed, permeabilized, and prepared for confocal microscopy using rabbit antioccludin, anticlaudin, anti-JAM-A, and anti-ZO-1antibodies and relevant secondary antibodies. All experiments were performed in triplicate, and representative images are shown. The scale bars correspond to 20 μm. (B) The internalized TJ molecules, shown as a percentage of intracellular fluorescence/total fluorescence, are presented as means ± standard errors from three independent experiments. Differences were evaluated using a one-way ANOVA. ***, P < 0.0001.

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  FIG 12. Inhibition of the RhoA/ROCK/MLC pathway restores the gate and fence functions of TJs in PSaV-infected polarized epithelial cells. Confluent LLC-PK cells grown on Transwell filters were untreated or treated with RhoA inhibitor CT04 (2 μg), ROCK inhibitor Y27632 (20 μM), or MLC inhibitor blebbistatin (10 μM) for 1 h at 37°C and then mock inoculated or inoculated with PSaV Cowden strain (MOI of 50) in the presence (A) or absence (B) of GCDCA for 5, 30, or 90 min. (A and B) Paracellular flux of FD4 and FD70 was measured in the apical-to-basolateral direction. The amount of FITC-dextran diffused to the basolateral side of the monolayer was normalized to the average obtained from control LLC-PK cells. As a positive control, confluent LLC-PK cells were treated for 10 min with 1.8 mM EGTA. All experiments were performed in triplicate. Data are presented as means ± standard errors from three independent experiments. Differences were evaluated using a one-way ANOVA. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. (C and D) The distribution of the membrane fluorescent marker BodipyFL-C12-sphingomyelin-BSA (5 nmol/ml) loaded onto the apical surface of LLC-PK cells was determined via…