Figure 5

HSP70 regulates DHAV-1 translation through interaction with the IRES. A DEFs were co-transfected with Rluc-IRES-Fluc and HSP70-HA. After 24 h, the samples were harvested for dual-luciferase assays. B DEFs were transfected with siRNA-HSP70 or siRNA-NC and then transfected with Rluc-IRES-Fluc after 36 h. Samples were collected at 18 h post-transfection, and luciferase activity was measured. C DEFs were transfected with HSP70-HA, and the cells were infected with DHAV-1 at 24 h post-transfection. The cell lysates were collected, and a mouse anti-HA antibody or mouse anti-IgG was added. The pulled down RNAs were extracted and amplified via RT‒PCR using DHAV-1 IRES primers. D DEFs were transfected with HSP70-HA. After 24 h, the cell lysates were incubated with biotinylated or nonbiotinylated IRES, RNA-binding proteins were collected, and their expression was analyzed via Western blotting. E Schematic diagram of Hsp70 truncation mutants. F, G HA-tagged truncated forms of HSP70, i.e., HSP70(1-384)-HA and HSP70(384-634)-HA, were co-transfected into cells with Rluc-IRES-Fluc, and luciferase activity was measured. H, I HA-tagged truncated forms of HSP70 were incubated with biotinylated or nonbiotinylated IRES and analyzed via an RNA pulldown assay. **, P < 0.01; ***, P < 0.001.