Methods for studying IRES-mediated translation of positive-strand RNA viruses

Internal ribosome entry sites are RNA elements that mediate translation in a cap-independent manner. A subset of positive strand RNA viruses utilize an IRES mechanism as a viral strategy to ensure efficient viral protein synthesis. IRES elements vary in sequence, structure, and factor requirements between virus families. Here, we describe methods to determine IRES activity and approaches to study the regulation and function of IRES-mediated translation both in vitro and in vivo. Finally, we describe a new IRES-directed reporter system which exploits the 2A ‘self-cleavage’ or ‘stop–go’ peptide for optimal detection of IRES activity.