Mapping of secondary structure of the spacer region within the 5'-untranslated region of the Coxsackievirus B3 RNA: possible role of an apical GAGA loop in binding La protein and influencing internal initiation of translation

Abstract

Translation initiation of the coxsackievirus B3 (CVB3) RNA has been shown to be mediated by a highly ordered structure of the 5'-UTR, which harbors an internal ribosome entry site (IRES). In this study, we have investigated the 48S ribosome assembly site and also characterized the intervening spacer region between the cryptic AUG₅₉₁ and the initiator AUG₇₄₂. The ribosomal complex formation was mapped by toe-printing experiment using rabbit reticulocyte lysate, which showed a major toe print at nucleotide U570 corresponding to the 48S ribosome assembly site at the putative Shine-Dalgarno like sequence. Elucidation of the secondary structure of a segment encompassing the ribosome binding site and the downstream spacer region by nuclease probing and chemical modifications demonstrated distinct stem and loop structure. Interestingly, a GAGA loop in the apical region of stem-loop H was found to be phylogenetically conserved as a GNRA-loop among the coxsackie B viruses. Deletion or substitution mutation of this apical GAGA loop drastically reduced binding with human La protein and significantly affected the IRES function. The study revealed important insights into the possible role of the intervening spacer region in cellular protein binding and influencing internal initiation of translation of CVB3 RNA.