The 2A proteinase (2Apro) of human rhinovirus 2 is a cysteine proteinase with a unique chymotrypsin-like fold. During viral replication, 2Apro performs self-processing by cleaving between its own N terminus and the C terminus of the preceding protein, VP1. Subsequently, 2Apro cleaves the two isoforms of the cellular protein, eukaryotic initiation factor (eIF) 4G. We have previously shown that HRV2 2Apro can directly bind to eIF4G isoforms. Here we demonstrate using deletion mutants of eIF4GI that HRV2 2Apro requires eIF4GI amino acids 600-674 for binding; however, the amino acids at the cleavage site, Arg681 downward arrow Gly, are not required. The HRV2 2Apro binding domain for eIF4GI was identified by site-directed mutagenesis. Specifically, mutations Leu17 --> Arg and Asp35 --> Glu severely impaired HRV2 2Apro binding and thus processing of eIF4GI in rabbit reticulocyte lysates; self-processing, however, was not affected. Alanine scanning analysis further identified the loop containing residues Tyr32, Ser33, and Ser34 as important for eIF4GI binding. Although Asp35 is part of the catalytic triad, most of the eIF4GI binding domain lies in a unique exosite structure absent from other chymotrypsin-like enzymes and is distinct from the substrate binding cleft. The exosite represents a novel virulence determinant that may allow the development of specific inhibitors for HRV2 2Apro.