Fig. 3
From: DEAD box RNA helicase 5 is a new pro-viral host factor for Sindbis virus infection
DDX5 interacts with the SINV capsid protein. A Schematic illustration of SINV genomic organization. The 5´ cap and poly(A) tail are depicted. The non-structural proteins (np1, nsp2, nsp3, nsp4) from the first ORF (in green and light blue) and the structural proteins (capsid, E3, E2, 6K, E1) from the second ORF (in yellow and grey) are shown. The sequences coding for nsp1, nsP2, nsP3, nsP4 and capsid proteins were individually cloned and fused with an N-terminal FLAG-HA tag. B Western blot on total lysate (INPUT) or anti-HA IP (IP) from transiently transfected HCT116 cells using anti-FLAG and anti-DDX5 antibodies. Position and molecular weight (kDa) of protein size markers are shown on the left. C Western blot on total lysate (INPUT), IgG-IP (IgG), DDX5-IP (DDX5) or DDX17-IP (DDX17) samples in both mock and SINV-GFP infected lysates, using antibodies against DDX5, DDX17 and viral capsid. The non-specific signal observed in control and DDX5 IP, both in mock and SINV samples, is due to the residual background signal from the IgG used for the IP. D Representative agarose gel showing the total RNA before and after RNase treatment. E Western blot on total lysate (INPUT), IgG-IP (IgG) or DDX5-IP (DDX5) samples in both mock and SINV-GFP infected lysates, treated or not with RNase A, using antibodies against DDX5, DDX17 and viral capsid. F-G Confocal co-immunofluorescence analysis performed in mock and SINV- infected HCT116 cells using specific primary antibodies. Rabbit anti-capsid (in green) and mouse anti-DDX5 antibody (in magenta) are shown in (F). Rabbit anti-DDX5(in green) and mouse anti-DDX17 antibodies (in magenta) are shown in (G). DAPI was used to stain the nuclei (in blue). Scale bar 10 μm. Representative fluorescence intensity profiles of capsid (green curve) and DDX5 (magenta curve) (F) or DDX5 (green curve) and DDX17 (magenta curve) (G) along the yellow line represented on the merge panel and normalized to the maximum value are shown on the right. One representative experiment out of three is shown in (B and E-G)