Rab18 binds to classical swine fever virus NS5A and mediates viral replication and assembly in swine umbilical vein endothelial cells

Figures

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  Figure 1.. Knockdown of Rab18 inhibits CSFV production. (a–c) Knockdown of Rab18 in SUVECs by lentivirus-mediated shRNA interference. (a) Confirmation of NTshRNA, shRab18-1, shRab18-2, and shRab18-3 cell lines by detection of the enhanced green fluorescent protein (EGFP) reporter. (a) Mock-transfected SUVECs. (b) SUVECs transfected with lentiviruses expressing NTshRNA. SUVECs transfected with lentiviruses expressing (c) shRab18-1, (d) shRab18-2, or (e) shRab18-3. Scale bars, 100 μm. (b) qRT-PCR and western blot analyze Rab18 gene and protein expression levels in Rab18 knockdown cell lines. β-actin served as an internal control. (c) Cell viability of shRab18-3 cell lines. (d) qRT-PCR analyzes CSFV RNA level and TCID50L assay to detect CSFV viral titers in the supernatants of NTshRNA cell lines, shRab18-3 cell lines, and Rab18-mutant-transfected cells. (e) Western blot analyzes Rab18-mutant in transfected cells using an anti-Flag antibody. β-actin served as an internal control.

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  Figure 2.. Overexpression of Rab18 enhances CSFV production. (a–c) Overexpression of Rab18 in SUVECs. (a) Confirmation of CMV and CMV-Rab18 cell lines by detection of the enhanced green fluorescent protein (EGFP) reporter. (a) Mock-transfected SUVECs. SUVECs transfected with lentiviruses expressing (b) CMV or (c) CMV-Rab18. Scale bars, 100 μm. (b) qRT-PCR and western blot analyzes Rab18 gene and protein expression levels in Rab18 overexpression cell lines. β-actin served as an internal control. (c) Cell viability of CMV-Rab18 cell lines. (d) qRT-PCR analyzes CSFV RNA level and TCID50 assay to detect CSFV viral titers in the supernatants of CMV cell lines and CMV-Rab18 cell lines. (e) Western blot analyzes pCDNA-Flag-Red-Rab18, pCDNA-Flag-Red-Rab18 (Q67L), and pCDNA-Flag-Red-Rab18 (S22 N) in transfected cells by anti-Flag antibody. β-actin served as an internal control. (f) qRT-PCR analyzes CSFV RNA level and TCID50 assay to detect CSFV viral titers in supernatants of pCDNA-Flag-Red-Rab18-, pCDNA-Flag-Red-Rab18(Q67L)-, and pCDNA-Flag-Red-Rab18(S22N)-transfected SUVECs. β-actin served as an internal control.

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  Figure 3.. Rab18 is involved in CSFV replication and assembly steps. (a) (a) In the virus binding assay, NTshRNA and shRab18-3 cell lines were incubated with CSFV for 1 h at 4ºC, total cell RNA was extracted, and the CSFV RNA level was quantified by qRT-PCR. β-actin served as an internal control. In the virus entry assay, NTshRNA and shRab18-3 cell lines were incubated with CSFV for 1 h at 4ºC. Then, cells were washed with PBS and cultured in fresh medium at 37ºC for 2 h. Finally, total cell RNA was extracted, and the CSFV RNA level was quantified by qRT-PCR. β-actin served as an internal control. (b) Detection of CSFV entry by IFA. NTshRNA and shRab18-3 cell lines were infected with CSFV (5 MOI) for 1 h at 4ºC. Then, cells were washed with PBS and cultured in fresh medium at 37ºC for 2 h. Finally, cells were fixed with 4% paraformaldehyde, and immunofluorescence staining was performed using anti-E2 antibodies. Cells were counterstained with DAPI to label nuclei (blue). (b) Rab18 involvement in CSFV replication. (a) NTshRNA and shRab18-3 cell lines were incubated with CSFV at different time points (1, 3, 5, 7 h), total cell RNA was extracted, and CSFV RNA level was quantified by qRT-PCR.…

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  Figure 4.. Rab18 binds to CSFV NS5A. (a) Co-immunoprecipitation (co-IP) assay. Cells were transfected with pcDNA-NS2-Myc or pcDNA-NS5A-Myc for 48 h. The transfected cells were lysed and immunoprecipitated, and western blot analysis was conducted using anti-Myc, anti-Rab18, and anti-β-actin. (b) GST-pulldown assay. GST or GST-Rab18 fusion proteins expressed in E. coli BL21 (DE3) were purified with glutathione agarose resin and incubated with the lysate of NS5A-Myc-expressing cells. Western blot analysis using anti-GST, anti-Myc, and anti-β-actin. (c) Exogenous NS5A-Myc binds to Rab18-Flag, S22N-Flag, and Q67 L-Flag in co-transfected cells. pcDNA-NS5A-Myc with pCDNA3.1-Rab18-Flag-, pCDNA3.1-S22N-Flag-, and pCDNA3.1-Q67L-Flag-transfected cells were lysed and immunoprecipitated. Then, western blot analysis was conducted using anti-Myc, anti-Flag, and anti-β-actin. pcDNA-NS2-Myc with pCDNA3.1-Rab18-Flag was used as a negative control. (d) Rab18 co-localization with CSFV NS5A protein. Cells were co-transfected with NS5A-GFP and Rab18-Red, NS5A-GFP and Q67L-Red, and NS5A-GFP and S22N-Red. Plasmids pEGFP-N1 and pCDNA-Red were co-transfected as a control. At 48 h after transfection, cells were fixed…

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  Figure 5.. Redistribution of NS5A in Rab18 knockdown cells. (a) Rab18 localization at the Golgi apparatus. Cells seeded on glass coverslips were transfected with a Rab18-Red, Rab18 (Q67L)-Red, or Rab18 (S22N)-Red expression plasmid, as indicated. At 48 h after transfection, cells were fixed in 4% paraformaldehyde, and immunofluorescence staining was performed using an anti-TGN46 antibody. Cells were also counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm. (b) NS5A distribution in a punctate pattern. NTshRNA and shRab18 cell lines seeded on glass coverslips were respectively transfected with pcDNA-Myc and NS5A-Myc expression plasmids, as indicated. At 48 h after transfection, cells were fixed in 4% paraformaldehyde, and immunofluorescence staining was performed using an anti-Myc antibody. Cells were also counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm.