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Multiple conserved structural cis-acting regulatory elements have been recognized both in the coding and untranslated regions (UTRs) of the hepatitis C virus (HCV) genome. For example, the cis-element 5BSL3.2 in the HCV-coding region has been predicted to use both its apical and internal loops to interact with the X RNA in the 3'-UTR, with the IIId domain in the 5'-UTR and with the Alt sequence in the coding region. Additionally, the X RNA region uses a palindromic sequence that overlaps the sequence required for the interaction with 5BSL3.2, to dimerize with another HCV genome. The ability of the 5BSL3.2 and X RNA regions to engage in multi-interactions suggests the existence of one or more molecular RNA switches which may regulate different steps of the HCV life cycle. In this study, we used biophysical methods to characterize the essential interactions of these HCV cis-elements at the molecular level. Our results indicate that X RNA interacts with 5BSL3.2 and another X RNA molecule by adopting two different conformations and that 5BSL3.2 engages simultaneously in kissing interactions using its apical and internal loops. Based on these results, we propose a mode of action for possible molecular switches involving the HCV RNA.

Hepatitis C virus RNA: molecular switches mediated by long-range RNA–RNA interactions?