FIG 6.

NSP3 binding to eIF4G stabilizes the eIF4E-eIF4G interaction. (A) Schematic representation of NSP3 fragments. Different fragments of NSP3 (RF strain) containing the RoXaN- and eIF4G-binding sites (cNSP3) or devoid of either the eIF4G- or RoXaN-binding domain (cΔ4G or cΔRX) were fused to GFP. RNA refers to the RNA-binding domain of NSP3, and the numbers refer to the NSP3 amino acid sequence. (B) HeLa cells were transiently transfected with the different GFP-tagged NSP3 fragments, and their relative expression and interaction with eIF4GI (direct interaction), eIF4E (indirect interaction through eIF4G), and PABP (no interaction) were visualized by Western blotting using the indicated antibodies either directly (input) or after immunoprecipitation with anti-GFP antibodies (IP anti-GFP). NT, nontransfected; −Ab, IP without anti-GFP antibodies; *, nonspecific signal. The results are representative of three separate experiments. (C) eIF4E-eIF4GI interaction in HeLa cells transiently transfected with the different GFP-tagged NSP3 fragments was visualized by Western blotting after IP using either anti-eIF4GI (left) or anti-eIF4E (right) antibodies. Coimmunoprecipitated PABP also was visualized. Input, direct Western blotting; NT, nontransfected; −Ab, IP without any antibodies. The results are representative of three separate experiments.