Abstract
RNAs of many positive strand RNA viruses lack a 5' cap structure and instead rely on cap-independent translation elements (CITEs) to facilitate efficient translation initiation. The mechanisms by which these RNAs recruit ribosomes are poorly understood, and for many viruses the CITE is unknown. Here we identify the first CITE of an umbravirus in the 3'-untranslated region of pea enation mosaic virus RNA 2. Chemical and enzymatic probing of the approximately 100-nucleotide PEMV RNA 2 CITE (PTE), and mutagenesis revealed that it forms a long, bulged helix that branches into two short stem-loops, with a possible pseudoknot interaction between a C-rich bulge at the branch point and a G-rich bulge in the main helix. The PTE inhibited translation in trans, and addition of eIF4F, but not eIFiso4F, restored translation. Filter binding assays revealed that the PTE binds eIF4F and its eIF4E subunit with high affinity. Tight binding required an intact cap-binding pocket in eIF4E. Among many PTE mutants, there was a strong correlation between PTE-eIF4E binding affinity and ability to stimulate cap-independent translation. We conclude that the PTE recruits eIF4F by binding eIF4E. The PTE represents a different class of translation enhancer element, as defined by its structure and ability to bind eIF4E in the absence of an m(7)G cap.
Highlights
The RNAs of many positive sense RNA viruses contain a cap-independent translation element (CITE)3 that allows efficient translation in the absence of a 5Ј cap structure [11,12,13]
The 3Ј-UTR of PEMV RNA 2 Is Required for Cap-independent Translation Initiation and Ribosomal Frameshifting—Nothing is known about cap-independent translation of umbraviruses
Secondary Structure but Not the Sequence of the Branching Stem-loops Is Necessary for PEMV RNA CITE (PTE) Activity—To define the primary and secondary structures required for PTE function, mutations were introduced into the PTE of luciferase reporter construct pP2lucP2 containing the full-length 3Ј-UTR, and the resulting RNA transcripts were translated in wheat germ extract (Fig. 4A)
Summary
Plasmid Constructs and RNA Preparation by in Vitro Transcription—pPER2 is a plasmid containing PEMV RNA 2 cDNA [38]. The reaction was incubated at room temperature for 15 min and filtered through nitrocellulose and Hybond Nϩϩ nylon membranes (Amersham Biosciences), which were presoaked in washing buffer (25 mM HEPES-KOH, pH 7.6, 100 mM KCl, 4 mM MgCl2, 1 mM DTT, and 10% glycerol), the membranes were washed with 3 ϫ 100 l washing buffer. Both membranes were dried and exposed to a PhosphorImager screen. Each sample was measured in triplicate, and the data were obtained from three experiments
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