RSAD2 suppresses viral replication by interacting with the Senecavirus A 2 C protein

Figures

• 

  Figure 1. SVA infection reduced the expression levels of RSAD2 in BHK-21 and PK-15 cells. A and B The extracted proteins and RSAD2 mRNA from SVA- or mock-infected BHK-21 cells at various time points were analysed by western blotting with anti-RSAD2, anti-VP1, and anti-β-actin antibodies (A) and reverse transcription‒quantitative polymerase chain reaction (RT-qPCR) (B), respectively. C BHK-21 cells were infected with SVA or UV-inactivated SVA for 12 h and then processed and analysed as described in panel (A) D and E Proteins (D) and RSAD2 mRNA (E) from PK-15 cells infected with SVA were analysed as described in panels A and (B) The data are expressed as the means ± standard deviations (SDs) from three independent experiments (*P < 0.05; **P < 0.01; ****P < 0.0001).

• 

  Figure 2. RSAD2 inhibited SVA replication. A and B BHK-21 cells transfected with Flag-RSAD2 or Flag plasmids were infected with SVA for 6–12 h, and the extracted proteins (A) and whole-cell culture medium (B) were then analysed. C and D BHK-21 cells transfected with Flag-RSAD2 or Flag plasmids were infected with rSVA-eGFP, and the number of rSVA-eGFP-infected cells was determined by an indirect fluorescence assay (IFA). C, and the results are presented in a histogram (D). E and F PK-15 cells transfected with Flag-RSAD2 or Flag plasmids were infected with SVA for 6–12 h, and the extracted proteins (E) and whole-cell culture media (F) were then analysed. G and H PK-15 cells transfected with Flag-RSAD2 or Flag plasmids were infected with rSVA-eGFP for 12 h, and the number of rSVA-eGFP-infected cells was then determined by IFA (G). The results are presented in a histogram (H). I and L Effects of RSAD2 silencing in BHK-21 (I) and PK-15 cells (L). J, K, M, and N BHK-21 and PK-15 cells transfected with siRSAD2 or siCon were infected with SVA for 6–12 h, and the extracted proteins (J and M) and whole-cell culture media (K and N) were then analysed. The data are expressed as the means ± SDs from…

• 

  Figure 3. RSAD2 interacted with SVA 2 C. A BHK-21 cells were co-transfected with Flag-RSAD2 and various viral proteins linked with a GFP tag for 24 h, followed by fixation and incubation with anti-Flag antibodies (red signals) and DAPI (blue signals). The colocalization of RSAD2 and viral proteins was observed using confocal immunofluorescence microscopy. Scale bar, 20 μm. B HEK-293T cells co-expressing Flag-RSAD2 and GFP, GFP-L, GFP-VP1, GFP-VP2, GFP-VP3, GFP-VP4, GFP-2B, GFP-2 C, GFP-3 A, GFP-3 C, or GFP-3D for 24 h were lysed and analysed by co-IP. C and D HEK293T cells co-expressing GFP, GFP-2 C and/or Flag-RSAD2 for 24 h were lysed and immunoprecipitated with an anti-GFP or anti-Flag antibody, followed by co-IP analysis. E HEK-293T cells expressing GFP-2 C or GFP for 24 h were immunoprecipitated with an anti-GFP antibody, and endogenous RSAD2 was detected.

• 

  Figure 4. The aa 201–322 domain of SVA 2 C plays an important role in the interaction between RSAD2 and SVA 2 C.A Schematic representation of the lengths of various truncated SVA 2 C proteins. B HEK-293T cells co-expressing GFP, GFP-2 C, GFP-2 C (aa 1–200), or GFP-2 C (aa 201–322) and Flag-RSAD2 for 24 h were lysed, immunoprecipitated, and analysed via various antibodies. C HEK-293T cells co-expressing GFP, GFP-2 C (aa 101–322), or GFP-2 C (aa 201–200) and Flag-RSAD2 were analysed as described in panel A. D BHK-21 cells were co-transfected with pEGFP-C1, pEGFP-2 C (aa 1–200), pEGFP-2 C (aa 101–322), or pEGFP-2 C (aa 201–322) and Flag-RSAD2 plasmids and then processed as described in Figure 3A. Scale bar, 20 μm.

• 

  Figure 5. The aa 43–70 region of RSAD2 is required for RSAD2-mediated inhibition of SVA replication. A Schematic representation of various structural domains of RSAD2. B HEK-293T cells co-expressing Flag, Flag-RSAD2, Flag-RSAD2∆N, Flag-RSAD2∆SAM, or Flag-RSAD2∆C and GFP-2 C were lysed, immunoprecipitated with an anti-Flag antibody, and then analysed with various antibodies. C HEK-293T cells co-transfected with GFP or GFP-2 C and Flag-RSAD2 or Flag-RSAD2∆ERLD were processed, immunoprecipitated with an anti-GFP antibody, and then analysed as described in panel B. D HEK-293T cells co-transfected with GFP or GFP-2 C and Flag-RSAD2, Flag-RSAD2∆N, or Flag-RSAD2∆(aa 43–70) were processed, immunoprecipitated with an anti-GFP antibody, and then analysed as described in panel B. E BHK-21 cells were transfected as described in panel D, and the colocalization of SVA 2 C and RSAD2 mutants was observed. F and G BHK-21 cells transfected with Flag, Flag-RSAD2, Flag-RSAD2∆ (ERLD), or Flag-RSAD2∆ (aa 43–70) were infected with SVA for 12 h and then analysed for VP1 expression (F) and viral titres (G). H and I BHK-21 cells transfected with Flag-RSAD2 or its mutated plasmids were infected with rSVA-eGFP, and…

• 

  Figure 6. Role of IFN-I in regulating RSAD2 expression in PK-15 cells. A-C The levels of IFN-β (A) and RSAD2 (B) mRNA or RSAD2 protein (C) were analysed by qPCR or western blotting, respectively, in PK-15 cells treated with SeV in the presence or absence of ruxolitinib. D-F The levels of IFN-β (D) and RSAD2 (E) mRNA or RSAD2 protein (F) were analysed in poly (I: C)-transfected cells in the presence or absence of ruxolitinib. G‒J PK-15 cells treated with SeV or poly (I: C) were infected with SVA. The expression levels were then determined via anti-VP1, anti-RSAD2, or anti-β-actin antibodies (G and I), and the viral titres were subsequently calculated (H and J). K–N PK-15 cells treated with SeV or poly (I: C) were infected with SVA, followed by treatment with ruxolitinib. The expression levels were then determined via anti-VP1, anti-RSAD2, or anti-β-actin antibodies (K and M), and viral titres were calculated (L and N). O Effect of JAK1 silencing in PK-15 cells (O). P and Q PK-15 cells transfected with siJAK1 or siCon and treated with poly (I: C) were infected with SVA for 6–12 h, and the extracted proteins (P) and whole-cell culture media (Q) were then analysed. R and S PK-15 cells were…