Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

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  FIG 1. PSaV-induced early activation of PI3K/Akt and MEK/ERK signaling pathways. (A and B) LLC-PK cells were inoculated with the PSaV Cowden strain (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C and then infected with or without PSaV in the presence of 200 μM GCDCA. Cell lysates were harvested at 5 min postinoculation (mpi). The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (D) LLC-PK cells transfected with scrambled siRNA (Scram) or siRNA against PI3K p85α or MEK were harvested at 24 and 48 h posttransfection. The downregulation of each protein by siRNA knockdown was evaluated by Western blotting using antibodies specific for each protein.…

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  FIG 2. Determination of chemical-mediated cytotoxicity in LLC-PK cells by 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 medium was thoroughly removed and replaced with 200 μl of MTT solution for 4 h at 37°C. Each well was incubated with 100 μl of DMSO for 10 min at room temperature. Cell viability was measured using an enzyme-linked immunosorbent assay (ELISA) reader to obtain the optical density at 570 nm.

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  FIG 3. Activation of PI3K/Akt and MEK/ERK signaling pathways by direct interaction of PSaV in the absence of GCDCA. (A and B) LLC-PK cells were incubated with PSaV (MOI of 1 FFU/cell) in the absence of GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C and D) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C (C) or transfected with or without siRNAs against PI3K p85α or MEK (D) and then infected with or without PSaV in the absence of GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (E and F) LLC-PK cells were incubated with PSaV VLPs (10 μg/ml), and the cell lysates were harvested at 5 mpi and prepared for Western blotting as described above. (G and H) LLC-PK cells were mock pretreated or…

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  FIG 4. Electron micrograph and Western blot analyses of PSaV Cowden strain VLPs. (A) PSaV VLPs harvested from recombinant baculovirus-infected Sf9 cells at 72 h postinfection were purified by CsCl density gradient ultracentrifugation and visualized by negative staining with 3% phosphotungstic acid (pH 7.0) under an electron microscope. The inset shows a higher magnification of the left panel. (B) Cell lysates of recombinant baculovirus-infected Sf9 cells at 72 h postinfection, their supernatants, and CsCl-purified VLPs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and VLPs were detected by Western blot analysis using anti-PSaV capsid hyperimmune antisera.

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  FIG 5. Bile acid-induced early activation of PI3K/Akt and MEK/ERK signaling pathways. (A and B) LLC-PK cells were treated with or without 200 μM GCDCA and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were determined by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C and then treated with or without 200 μM GCDCA. Cell lysates were harvested at 5 min posttreatment. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (D) LLC-PK cells were transfected with or without siRNAs against PI3K p85α or MEK and then treated with or without 200 μM GCDCA. Cell lysates were harvested at 5 min posttreatment. The expression levels of pAkt (Ser473), PI3K, pERK (Thr202/Tyr204), MEK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. The intensity of each…

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  FIG 6. Activation of PI3K/Akt and MEK/ERK signaling pathways by interaction of PSaV with cell surface carbohydrate receptors. (A and B) LLC-PK cells were infected with mock-labeled PSaV particles or Alexa Fluor 594 (AF594)-labeled PSaV particles and incubated for 36 h at 37°C in the presence of 200 μM GCDCA. The cells were then harvested by freezing and thawing, and the virus titers were determined by TCID50 assay (A) and the genome copy number by real-time RT-PCR (B) as described in Materials and Methods. (C) LLC-PK cells were treated with or without 1 or 5 mM sodium periodate (NaIO4) or 100 or 200 mU Vibrio cholerae neuraminidase (NA) to remove carbohydrates or sialic acids from the cell surface, respectively, incubated with AF594-labeled PSaV particles (approximately 415 particles per cell) for 30 min at 4°C in the absence of 200 μM GCDCA, and subsequently examined by confocal microscopy. This experiment was repeated three independent times, and one representative set of results is shown. Bars, 20 μm. (D and E) LLC-PK cells were treated with or without 1 or 5 mM NaIO4 or 200 mU NA for 1 h at 37°C (D) or treated with 1 or 20 mM MβCD for 1 h at 37°C or with 10 or 200 µM soluble…

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  FIG 7. Inhibition of PSaV trafficking by blockade of PI3K/Akt and MEK/ERK signaling pathways. (A) LLC-PK cells were incubated with Alexa Fluor 594 (AF594)-labeled PSaV particles (approximately 415 particles per cell) for the indicated times at 37°C in the presence of 200 μM GCDCA. The cells were then fixed, permeabilized, and further incubated with a monoclonal antibody against the early endosome marker EEA1 or the late endosome marker LAMP2. After incubation with a FITC-conjugated anti-mouse IgG antibody, the cells were processed for confocal microscopy to determine the colocalization of AF594-labeled PSaV particles with the early endosome marker EEA1 or the late endosome marker LAMP2. The boxed areas are magnified and shown under each panel. (B and C) LLC-PK cells were pretreated with or without wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) for 1 h at 37°C and then infected with AF594-labeled PSaV particles (approximately 415 particles per cell) in the presence (B) or absence (C) of 200 μM GCDCA for 3 h. After fixation and permeabilization, the cells were incubated with a monoclonal antibody against EEA1 or LAMP2 and then with a FITC-conjugated secondary antibody to visualize…

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  FIG 8. Inhibition of the PI3K/Akt and MEK/ERK signaling pathways affects PSaV infectivity and viral protein expression. (A to D) LLC-PK cells were pretreated with noncytotoxic concentrations of wortmannin or U0126 for 1 h at 37°C and then infected with PSaV (MOI of 1 FFU/cell) for 36 h in the presence (A and B) or absence (C and D) of 200 μM GCDCA. (A and C) Levels of PSaV VPg protein were determined by Western blotting. GAPDH was used as a loading control. The intensity of VPg relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane. (B and D) Viral titers were determined by TCID50 assay. The data are presented as means and standard deviations of the results of three independent experiments. Differences were evaluated using one-way analysis of variance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.00001.

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  FIG 9. Silencing of PI3K and MEK traps PSaV particles in early endosomes. (A and B) LLC-PK cells were transfected with scrambled siRNA or siRNA against PI3K p85α or MEK and then incubated with Alexa Fluor 594 (AF594)-labeled PSaV particles (approximately 415 particles per cell) for 3 h in the presence (A) or absence (B) of 200 μM GCDCA. After fixation and permeabilization, the cells were incubated with a monoclonal antibody against the early endosome marker EEA1 or the late endosome marker LAMP2 and then with a FITC-conjugated secondary antibody and processed for confocal microscopy to determine the colocalization of AF594-labeled PSaV particles with EEA1 or LAMP2. The boxed areas are magnified and shown under each panel. All experiments were performed in triplicate, and a representative set of results is shown. Bars, 20 μm. (C and D) Quantification of AF595-labeled PSaV particles colocalized with the early endosome marker EEA1 (C) and the late endosome marker LAMP2 (D) was performed using 10 confocal microscopy images of cells treated under the conditions described above by use of the ImageJ program. Quantification of signals was made with a threshold of 0.03 to 1.3 µm2 as described in…

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  FIG 10. V-ATPase-induced endosomal acidification is required for PSaV infection. (A and B) LLC-PK cells were pretreated with or without noncytotoxic concentrations of bafilomycin A1 for 1 h at 37°C and then infected with or without PSaV (MOI of 1 FFU/cell) for 36 h in the presence of 200 μM GCDCA. (A) Levels of PSaV VPg protein were determined by Western blotting. GAPDH was used as a loading control. The intensity of VPg relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane. (B) Viral titers were determined by TCID50 assay. All experiments were performed in triplicate, and a representative set of results is shown. The data are presented as means and standard deviations of the means of the results of three independent experiments. Differences were evaluated using one-way analysis of variance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.00001.

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  FIG 11. Coimmunoprecipitation (IP) of phosphorylated PI3K and ERK signaling molecules with the V-ATPase pump. (A to D) Serum-starved LLC-PK cells were inoculated with or without the PSaV Cowden strain (MOI of 1 FFU/cell) for the indicated times. Subsequently, the cell lysates were immunoprecipitated using antibodies specific for V1 subunit E of V-ATPase (A), pPI3K (B), pAkt (C), and pERK (D). The coimmunoprecipitated products were analyzed by Western blotting to detect pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and V1 subunit E by use of the relevant antibodies. GAPDH was used as a loading control.

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  FIG 12. Silencing of PI3K and MEK inhibits the coimmunoprecipitation of phosphorylated PI3K and ERK signaling molecules with the V-ATPase pump. (A and B) LLC-PK cells were transfected with scrambled siRNA or siRNA against PI3K p85α (A) or MEK (B) and then incubated with the PSaV Cowden strain (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA for the indicated times. Subsequently, the cell lysates were immunoprecipitated using antibodies specific for V1 subunit E of V-ATPase, pPI3K, pAkt, and pERK. The coimmunoprecipitated products were analyzed by Western blotting to detect pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and V1 subunit E by use of the relevant antibodies. GAPDH was used as a loading control.