A SINGLE NUCLEOTIDE SUBSTITUTION IN THE INTERNAL RIBOSOME ENTRY SITE OF FOOT-AND-MOUTH-DISEASE VIRUS LEADS TO ENHANCED CAP-INDEPENDENT TRANSLATION INVIVO

Mutants of foot-and-mouth disease virus (FMDV) with altered biological properties can be selected during the course of persistent infection of BHK-21 cells with FMDV C-S8c1 (J. C. de la Torre, E. Martinez-Salas , J. Diez, A. Villaverde, F. Gebauer, E. Rocha, M. Davila, and E. Domi ngo, J. Virol. 62:2050-2058, 1988). Two nucleotide substitutions, U to C at position -376 and A to G at position -15, (counting as +1 the A of the first functional AUG), were fixed within the internal ribosome entry site (IRES) of R100, the virus rescued after 100 passages of the carrier BHK-21 cells. IRES-directed cap-independent protein synthesis was quantitated by using bicistronic constructs of the form chloramph enicol acetyltransferase gene-IRES-luciferase gene. The IRES from R100 was 1.5- to 5-fold more active than that of C-S8cl in directing cap-i ndependent luciferase synthesis. This enhanced translational activity was observed when the RNAs were transcribed either in the nucleus or i n the cytoplasm by a weak or a strong promoter, respectively. C-S8cl a nd R100 IRES elements were functional in both FMDV-sensitive and FMDV- resistant cells (including persistently infected R cells), indicating that factors mediating cap-independent protein synthesis are not limit ed in any of the analyzed cell lines. Constructs in which each of the two mutations in the R100 IRES were analyzed separately indicate that the transition at position -376 is responsible for the enhanced activi ty of the R100 IRES. By estimating the effect that an increase in the initial translation efficiency may have on subsequent RNA replication steps, we suggest that the modifications in the IRES elements can acco unt for the previously described hypervirulence of FMDV R100 for BHK-2 1 cells. The results show that a single point mutation in an IRES elem ent of a picornavirus can cause an increase in translation efficiency.