A novel protein-RNA binding assay: Functional interactions of the foot-and-mouth disease virus internal ribosome entry site with cellular proteins

Translation initiation on foot-and-mouth disease virus (FMDV) RNA occurs by a cap-independent mechanism directed by a highly structured element (simil ar to 435 nt) termed an internal ribosome entry site (IRES). A functional a ssay to identify proteins that bind to the FMDV IRES and are necessary for FMDV IRES-mediated translation initiation has been developed. In vitro-tran scribed polyadenylated RNAs corresponding to the whole or part of the FMDV IRES were immobilized on oligo-dT Dynabeads and used to deplete rabbit reti culocyte lysate (RRL) of IRES-binding proteins. Translation initiation fact ors elF4G, elF4A, and elF4B bound to the 3' domain of the FMDV IRES. Deplet ion of elF4G from RRL by this region of the FMDV IRES correlated with the l oss of translational capacity of the RRL for capped, uncapped, and FMDV IRE S-dependent mRNAs. However, this depleted RRL still supported hepatitis C v irus IRES-directed translation. Poly (rC) binding protein-2 bound to the ce ntral domain of the FMDV IRES, but depletion of RRL with this IRES domain h ad no effect on FMDV IRES-directed translation initiation.