An Apical GAGA Loop within 5’ UTR of the Coxsackievirus B3 RNA Maintains Structural Organization of the IRES Element Required for Efficient Ribosome Entry

Abstract

Coxsackievirus B3 (CVB3) a close relative of Poliovirus has been implicated as the primary cause of dilated cardiomyopathy in humans. The single-stranded, plus sense, viral genomic RNA is naturally uncapped and the translation of viral encoded protein is initiated from an ‘Internal Ribosome entry site’ (IRES) element at the 5' untranslated region (UTR). The 741 nucleotide 5'UTR is highly structured, with defined stem loops. Previously, we have reported results of probing the secondary structure of the 5'UTR RNA, and showed the existence of 11 putative stem loop (SL) structures, SL A-K. Further, using mutants, we showed the importance of an apical GAGA loop in SL-H, in IRES activity. In addition to significantly reducing CVB3 IRES activity the mutations in the 5'UTR drastically reduced interaction with La autoantigen, a critical trans acting factor. Here, we demonstrate that mutation of the GAGA loop to CAGU alters the structure around SL-H, and reduces the interaction of La protein with critical cis acting elements of the IRES RNA. Interestingly, mutation of the loop to GAAA also has similar effect, suggesting the GAGA sequence of the loop, is necessary for the function. Furthermore, using gene specific siRNA against La protein we have demonstrated La protein to be a bona fide trans acting factor for CVB3 IRES. However, upon partial knockdown of La protein, translation mediated by the mutant 5'UTRs are affected to a similar extent as the wild type, implicating the structural alteration as the reason for the effect of the mutations on IRES activity.