Figure 4

PCBP2 regulates DHAV-1 translation through interaction with the IRES. A Schematic diagrams of the bicistronic reporter plasmids CMV-RHF and CMV-RHF-IRES. B The effects of PCBP2 on DHAV-1 IRES activity were determined via bicistronic reporter plasmids. DEFs in which PCBP2 was overexpressed or knocked down were transfected with the CMV-RHF or CMV-RHF-IRES plasmid. Twenty-four hours post-transfection, the activities of the FLuc and RLuc reporters were measured. The bars in the histogram indicate FLuc/RLuc activity as percentages. The experiments were performed in triplicate, and the results are presented in the bar graph. The expression levels of PCBP2, 3 × Flag-PCBP2, and actin were measured by western blotting. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. C DEFs were transfected with 3 × Flag-PCBP2 and were then infected with DHAV-1 at 12 h post-transfection. The cell lysates were collected, and a mouse anti-HA antibody or mouse IgG was added. The pulled down RNAs were extracted and amplified via PCR using DHAV-1 IRES primers. D DHAV-1 IRES RNA was pulled down with purified PCBP2 or from cell lysates transfected with the 3 × Flag-PCBP2 plasmid. The 6 × His-tagged PCBP2 protein was purified by Ni affinity chromatography and then used for pulldown assays. Purified PCBP2 or cell lysates were incubated with the biotinylated or nonbiotinylated IRES, RNA-binding proteins were collected, and their expression was analysed via western blotting.