Mutational analysis of the J-K stem-loop region of the encephalomyocarditis virus IRES

Cap-independent translation of encephalomyocarditis virus (EMCV) RNA is controlled by a segment of the 5' untranslated region termed the internal ribosomal entry site, or IRES. The IRES contains a series of stem-loop structural elements. The J and K stems (EMCV bases 682 to 795), near the center of the IRES, are well conserved among all cardio-, aphtho-, and hepatoviruses. We have examined the biological roles of these elements by constructing mutations within the J-K sequences of EMCV and testing the mutations for activity in translation, translation competition, UV cross-linking, and viral infectivity assays. Mutations near the helical junction of J and K proved severely detrimental to both cellular translation and cell-free translation of downstream cistrons. The same mutations reduced the ability of the IRES to compete for cellular factors in competition assays and reduced the infectivity of viral genomes carrying these lesions. A mutation in the terminal loop of J gave similar results. In contrast, mutations within the terminal loop of K had minimal impact on in vitro translation activity and IRES competitive ability. However, in vivo analysis of the K-loop mutations revealed deficiencies during cellular translation and further showed markedly reduced infectivity in HeLa cells. UV cross-linking experiments identified a 49-kDa protein which interacts strongly with the J-K region, but the identity of this protein and its contribution to IRES activity are unclear.