Fig. 3

FUBP3 regulated the viral protein translation and viral RNA replication. To establish the stable knocked down FUBP3 cell line, BHK-21 cells were transfected with shFUBP3 plasmid (5 µg) for 72 h, followed by selection with puromycin to obtain the FUBP3 stable knocked-down cells. The expression levels of FUBP3 was detected by Western blotting using anti-FUBP3 specific antibody. A β-actin expression was used as an internal control (a). The untreated BHK-21 cells (c) and sh-FUBP3-transfected cells (b) were then infected with JEV (MOI = 2) for 4, 8, 16, 24, 32, 40, 48 h and protein expression was analyzed by Western blotting using anti-NS specific antibody. A β -actin expression was used as an internal control. d Measurement of JE viral titers at 24 h post-infection from untreated and sh-FUBP3-transfected cells were performed by plaque forming assay. e The pCMV-FUBP3-Flag vector and pCMV-Flag vector (8 µg) were then transfected in the stable knocked-down FBP3 cells for 24 h, followed by infection with JEV (MOI = 2) for 4, 8, 16, 24, 32, 40, 48 h. The intracellular viral RNA production was then analyzed by RT-qPCR using specific primers as described in the Methods. (*p < 0.05; **p < 0.01; ***p < 0.001)