Uncovering the biophysical mechanisms of riboregulatory interactions involving the hepatitis C virus 3'X RNA using single-molecule FRET.

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The last 98 nucleotides of the hepatitis C virus genome form what is called the 3ʹX RNA, which is a structured and highly conserved non-coding RNA element that has been implicated in the regulation of several different RNA-dependent viral processes like replication, translation, and particle assembly. Current research suggests that this riboregulatory behavior arises from the ability of 3ʹX to form multiple mutually exclusive interactions with highly conserved RNA elements across the HCV genome. Here, we use time-resolved single-molecule fluorescence spectroscopy in conjunction with analytical size exclusion HPLC to characterize the structural, energetic, and kinetic principles governing these RNA-RNA interactions. In the absence of any interaction partners, we see that 3ʹX adopts two distinct conformations. Furthermore, these two conformations have different preferences for various inter- and intragenomic RNA-RNA interactions, with the interactions themselves depending on various external factors (e.g., [Mg2+]). These findings have made it possible for us to predict which of these mutually exclusive interactions will be formed under specific conditions and ultimately how the formation and disruption of them regulates various RNA-dependent viral processes.