IDENTIFICATION OF AN ESSENTIAL REGION FOR INTERNAL INITIATION OF TRANSLATION IN THE APHTHOVIRUS INTERNAL RIBOSOME ENTRY SITE AND IMPLICATIONS FOR VIRAL EVOLUTION

Translation of aphthovirus RNA is initiated at an internal ribosome en try site (IRES) element, preceding the first functional AUG initiation codon, The effect of mutations at the base of domain 3 of the aphthov irus IRES on translation activity has been analyzed by site-directed m utagenesis and expression of bicistronic RNAs in transfected cells, Th e results have shown that the enhanced IRES activity associated with a single pyrimidine transition fixed in a persistent aphthovirus varian t (E. Martinez-Salas, J. C. Saiz, M. Davila, G. J. Belsham, and E. Dom ingo, J. Virol. 67:3748-3755, 1993) is base specific. Mutations predic ted to destabilize the base of domain 3 were detrimental to IRES funct ion, but subsequent restoration of the RNA structure gave rise to full y competent IRES, In contrast, single or multiple mutations that did n ot affect predicted helical structures modified the relative efficienc y of translation by at most 10-fold, suggesting that primary sequence also plays a role in IRES activity. A correlation between the energy o f stabilization of the IRES structure and the efficiency of translatio n has been noted, None of the 15 mutations studied reached a level of initiation of translation comparable to that of the IRES from the pers istent variant, The results indicate a critical participation of the b ase of domain 3 in the activity of the aphthovirus IRES, with a strong effect of secondary or higher-order structures and minor effects of p rimary structure.