FIG 3.

Mapping of the HSC70-binding domain on NS5A protein. (A) Schematic representation of the HCV NS5A protein. NS5A consists of three domains (domains I, II, and III) separated by low-complexity sequences (LCS I and II). The position of the amino-terminal amphipathic helix membrane anchor is shown as “AH.” The NS5A deletion mutants contain amino acids of NS5A as indicated. Each NS5A deletion mutant contains either the HA tag in the N terminus or the myc-His₆ tag in the C terminus. The dark gray region represents the HA tag sequence. The light gray region represents the myc-His₆ tag. Closed boxes represent proteins that bound specifically to HSC70 protein, and open boxes represent those that did not bind. (B, C) Huh-7.5 cells were transfected with each NS5A mutant plasmid together with pCAG-FLAG-HSC70. At 48 h posttransfection, the cells were harvested. The cell lysates were immunoprecipitated with anti-FLAG beads, followed by immunoblotting with rabbit anti-HA PAb (B, upper panel, right) or anti-c-myc PAb (C, upper panel, right). Input samples were immunoblotted with rabbit anti-HA PAb (B, upper panel, left), anti-c-myc PAb (C, upper panel, left), or anti-FLAG MAb (B and C, bottom panels).