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The Research interests of our laboratory comprise all aspects of molecular biology of picornaviruses including RNA genome translation and proteolytic processing, viral RNA replication and folding, viral pathogenesis, bioinformatics, and sequence analysis. Among our lab's major goals are to explore and define the relationship of the cardiovirus genus to other members of the picornavirus family and to exploit the unique features of cardioviruses to learn about the molecular biology of this family. We have developed extensive panels of infectious cDNAs for encephalomyocarditis virus (EMCV) and Mengo, and we use high-tech recombinant engineering, reverse genetics and cell-free proteing synthesis assays to unravel virus life cycle, step-by-step. For the past several years our major research focus has been to study virus-host interacions, particularly the mechanisms of virus-induced shut off of host transcription, nucleocytoplasmic transport and cap-dependent translation.
vrology.wisc.edu/acp
The Technique
The methodology described in this article derives from the lab's research goals on cardioviral genome functions. We cloned and characterized the original pE5LVPO cDNA and distributed this plasmid as the founder material for all subsequent commercial and research applications on the internal ribosome entry site (IRES) from EMCV. This IRES and its various iterations are now widely used for the expression of eukaryotic proteins. Consequently, we field a great many inquiries about sequence differences and a great many inquiries about sequence differences and passage derivatives that may affect specific applications, especially when “the IRES doesn't work for us.” This article traces the history of commercial IRES variants and the reasons behind their variations. The data then compare protein expression from the most common IRESs, head-to-head in cells, and cell-free extracts, using bicistronic vectors. As expected, the presence of a native A6 bifurcation sequence in the J–K structural component of the IRES, combined with an authentic EMCV AUG codon context, provided maximal translational efficiency. Pertubation of one or both of these components is pervasive in many unsuccessful experiments. We hope this article clarifies these important differences and reinforces within the biotech community that the sequence, context, and history of a selected IRES can have profound impact on experimental outcomes.
Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location, p. 283.
