Figure 3.

Localization in Grb2 of the regions of interaction with NS5A. (A) Yeast two-hybrid analysis of NS5A and Grb2 interaction. The Hf7c reporter strain was transformed with the indicated plasmids. The interaction between the two hybrid proteins is indicated by the induction of HIS3 expression (growth on SD medium −His). NS5A-PKR K296R was used as a positive control (15). SH3-N and SH2-C represent the depicted Grb2 deletion mutants (Upper). AD denotes fusion with activation domain of GAL4; BD, fusion with DNA-binding domain of GAL4. Double transformants were plated on SD medium lacking Trp and Leu, and then colonies were patched on SD medium lacking Trp/Leu/His. (B) In vitro binding analysis. Purified recombinant NS5A was incubated with purified GST-Grb2 or GST-Grb2 mutants coupled to glutathione-agarose beads and precipitated as described in Fig. 2. A schematic representation of Grb2 and mutants is shown, but not to scale (Left). Mutant domains in which binding activity has been abrogated are indicated by Xs. Precipitated proteins were detected by immunoblotting by using anti-NS5A (Right). Amino acid is denoted by standard one-letter code. The position of NS5A is indicated by the arrow.