Interaction of Structural Glycoprotein E2 of Classical Swine Fever Virus with Protein Phosphatase 1 Catalytic Subunit Beta (PPP1CB)

Figure 1. Co-immunoprecipitation of E2 in CSFV-infected or mock-infected cells. The input cell lysate is in lanes 1 and 2 and the lysate that was immunoprecipitated with antibodies for E2 in lanes 3 and 4. Protein extracts were blotted for PPP1BC and E2 as indicated in Materials and Methods with bands observed at the expected molecular weights of 55kDa for E2 and 37 kDa for PPP1CB.

Figure 2. PLA for E2 and PPP1CB in CSFV infected SK6 cells. Interaction between E2 and PPP1CB was determined by PLA in SK6 cells that were either mock-infected or infected for 24 h with CSFV BICv (MOI = 10). Scale bar is 50 µM.

Figure 3. Prediction of phosphorylation sites in CSFV E2 (A) Amino acid sequence of CSFV E2 with serine displayed in red and threonine displayed in green. Highlighted residues indicate residues that are predicted to be phosphorylated. (B) Prediction in NetPhos displaying the phosphorylation potential.

Figure 4. Detection of E2 phosphorylation by PLA. Phosphorylation of E2 was determined with PLA using a pan phosphor-serine/threonine and E2 antibody in SK6 cells either mock infected or with CSFV BICv (MOI = 10). Scale bar is 50 µM.

Figure 5. Effect of PPP1CB phosphatase activity on CSFV replication. SK6 cells were pretreated with no drug, PPP1CB phosphatase inhibitor Okadaic Acid (OA), phosphatase activator Ceramide (C6) or no treatment (NT) followed by infection with CSFV (MOI:1) or mock for 2 h. Time points were collected 0 and 24 h post infection (hpi). (A) Viral yields from CSFV infection in SK6 cells treated as indicated. Titers were determined in SK6 cells and expressed as the log 10 TCID 50 /mL, +/−standard error from triplicate samples (OA: Okadaic Acid, C6: Ceramide) ** p Value: <0.01). (B) Whole-cell lysates were collected 24 hpi. Immunoblotting with an E2-specific antibody (band is 55 kDa as expected) showed a decrease in E2 protein concentration with C6 treatment compared to OA and NT (lane 6 vs. 4 and 5). An α-Tubulin-specific antibody was used as a loading control (band is at 50 kDa as expected).

Figure 6. Effect of PPP1CB-specific siRNA treatment on CSFV replication. SK6 cells were transfected with either siRNA targeting PPP1CB, a control siRNA (siCtrl), lipofectamine alone or left untreated (mock). Cells were infected with CSFV MOI 0.1 (A) Viral yields from CSFV infected SK6 cells treated as indicated. Supernatants were collected at 0 and 24 hpi and titrated in SK6 cells and expressed as log10 TCID50/mL +/−standard error from triplicate samples. (B) Whole-cell lysates were collected 48 h after siRNA treatment and analyzed by immunoblotting with a PPP1CB-specific antibody (band is 37 kDa) or with an α-Tubulin-specific antibody (band is at 50 kDa).