UC San Diego

The Encephalomyocarditis virus (EMCV) is a picornavirus that infects a broad range of mammals, including rodents, pigs, primates, and humans. Though the EMCV is not considered a significant source of human disease, it is deadly to many other mammals and has caused serious outbreaks in farms, zoos, and animal research facilities. Like the closely related polio virus (PV) and foot and mouth disease virus (FMDV), the EMCV genomic RNA contains an internal ribosome entry site (IRES) within its 5’ UTR that facilitates cap-independent translation initiation. The EMCV IRES is one of the most efficient IRES sequences and is therefore used extensively in biotechnology to enable cap-independent translation initiation. Despite its widespread use, the structure and mechanism of the EMCV IRES have yet to be established. The structure of the full length EMCV IRES has not yet been determined using structural methods. Though determination of the IRES structure by cryo-EM would provide valuable insight into the mechanism of IRES-driven translation initiation, structural studies of the IRES are challenging due to its flexible nature and high initiation factor requirements. In order to study the EMCV IRES using cryo-EM, we have developed a novel method to purified IRES initiation complexes from rabbit reticulocyte lysate (RRL) systems. With this method, we have isolated EMCV IRES-ribosome complexes and obtained a low-resolution structure of the IRES bound to the 40S ribosome. Our results indicate that our method is effective for purifying challenging mRNA complexes and could be used to obtain a high-resolution structure of the EMCV IRES complex in the future. The secondary structure of the EMCV IRES has been predicted by serval previous studies. Though certain domains of the IRES are consistent across the previous secondary structure predictions, the structure of the IRES I domain is highly variable from one prediction to the next. Using mutational analysis, we determined two helical regions within the I domain that are vital for IRES translation. The importance of these helical regions revealed that only one of the previous secondary structure predictions aligns with our mutational analysis. We also tested the SHAPE reactivity of the IRES before and after in vitro translation in RRL, and used this data to determine which regions of the IRES structure are protected from SHAPE reactivity during translation complex formation. The combination of these data allowed us to refine the EMCV IRES secondary structure and provided insight into the functional significance of the IRES I domain.