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Conserved Nucleotides within the J Domain of the Encephalomyocarditis Virus Internal Ribosome Entry Site Are Required for Activity and for Interaction with eIF4G

Angela T. Clark,^1, Morwenna E. M. Robertson,^1, Graeme L. Conn,² and Graham J. Belsham^1*

BBSRC Institute for Animal Health, Pirbright, Woking, Surrey GU24 ONF,¹ Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester M60 1QD, United Kingdom²

Received 24 February 2003/ Accepted 22 August 2003

The internal ribosome entry site (IRES) elements of cardioviruses (e.g., encephalomyocarditis virus [EMCV] and foot-and-mouth disease virus) are predicted to have very similar secondary structures. Among these complex RNA structures there is only rather limited complete sequence conservation. Within the J domain of the EMCV IRES there are four highly conserved nucleotides (A704, C705, G723, and A724)., which are predicted to be unpaired and have been targeted for mutagenesis. Using an IRES-dependent cell selection system, we have isolated functional IRES elements from a pool of up to 256 mutants. All changes to these conserved nucleotides resulted in IRES elements that were less efficient at directing internal initiation of translation than the wild-type element, and even some of the single point mutants were highly defective. Each of the mutations adversely affected the ability of the RNAs to interact with the translation initiation factor eIF4G.

Present address: Food Standards Agency, London WC2B 6NH, United Kingdom.

Present address: Department for the Environment, Food and Rural Affairs, London SW1E 6DE, United Kingdom.

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