With over 200 million people infected with hepatitis C virus (HCV) worldwide, there is a need for more effective and bettertolerated therapeutic strategies. The HCV genome is a positive-sense; single-stranded RNA encoding a large polyprotein cleaved at multiple sites to produce at least ten proteins, among them an error-prone RNA polymerase that confers a high mutation rate. Despite considerable overall sequence diversity, in the 39-untranslated region of the HCV genomic RNA there is a 98-nucleotide (nt) sequence named X RNA, the first 55 nt of which (X55 RNA) are 100% conserved among all HCV strains. The X55 region has been suggested to be responsible for in vitro dimerization of the genomic RNA in the presence of the viral core protein, although the mechanism by which this occurs is unknown. In this study, we analyzed the X55 region and characterized the mechanism by which it mediates HCV genomic RNA dimerization. Similar to a mechanism proposed previously for the human immunodeficiency 1 virus (HIV-1) genome, we show that dimerization of the HCV genome involves formation of a kissing complex intermediate, which is converted to a more stable extended duplex conformation in the presence of the core protein. Mutations in the dimer linkage sequence loop sequence that prevent RNA dimerization in vitro significantly reduced but did not completely ablate the ability of HCV RNA to replicate or produce infectious virus in transfected cells.

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.

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