Sequence alignment of human eIF4GI and eIF4GII proteins (sequence comparison starts from the eIF4E-binding site) (4, 18). Asterisks, positions of 2A^pro cleavage sites in eIF4GI and eIF4GII. The full-length eIF4GI and eIF4GII sequence accession numbers are AY082886 (3) and AF012072 (4), respectively.

(A) Identification of the 2A^pro cleavage site in eIF4GII. HeLa-I cells were infected with HRV16 (100 50% tissue culture infective doses per cell) as described previously (30). Cell extract (60 μg of protein; lane 1) was prepared 6 h postinfection and loaded on a gel in parallel with 40 μg of HeLa S10 (lane 2) or purified recombinant eIF4GII (1/40 of the reaction mixture, ∼0.5 μg; lane 3) that was incubated in the presence of purified HRV2 2A^pro. Proteins were resolved by SDS-8% PAGE and blotted onto nitrocellulose. The blot was treated with polyclonal antibodies against the cp_C of eIF4GII. WB, Western blot. (B and C) eIF4GII mutants that are resistant to HRV2 2A^pro cleavage. (B) Scheme of wild-type eIF4GII (1) and mutant (2 and 3) fragments. GST-eIF4GII (aa 445 to 744)-FLAG fragments contained a single point mutation (G700E) or a deletion (Δ674-702). (C) eIF4GII proteins were expressed in E. coli and purified on a glutathione-Sepharose column (4). Purified proteins (0.5 μg) were incubated in the presence or absence of recombinant 2A^pro (2.8 μg) in buffer containing 100 mM potassium acetate, 20 mM Tris-HCl (pH 7.6), 2.5 mM magnesium acetate, and 10% glycerol for 1 h at 30°C. Laemmli buffer was added to stop the reaction. Samples were resolved by SDS-PAGE (10.0, 15.5, or 12.5% [bottom] acrylamide). Proteins were transferred onto a nitrocellulose membrane, which was subsequently incubated with anti-GST or anti-FLAG antibodies as indicated. The small (62-aa) C-terminal cleavage product containing the FLAG epitope was resolved only in a 15.5% gel (cp_C). The wild-type eIF4GII fragment was cleaved by HRV2 or CVB4 2A^pro, as indicated. Positions of molecular mass standards are shown on the right.

Comparison of 2A^pro cleavage sites on the respective polyproteins and eIF4G homologues. The viral polyprotein sequences represent the C terminus of VP1 and the N terminus of 2A^pro, respectively, and are taken from the public database http://www.iah.bbsrc.ac.uk/virus/picornaviridae/sequencedatabase/index.html. The residues shown to be important for HRV2 2A^pro substrate recognition by peptide cleavage, mutational analysis, or crystallography are in boldface. P and P′ designate the N-terminal and the C-terminal cleavage products, respectively. Numbers indicate the positions of amino acid residues relative to the cleavage site.