Structural and functional analysis of the roles of the HCV 5' NCR miR122-dependent long-range association and SLVI in genome translation and replication.

The hepatitis C virus RNA genome possesses a variety of conserved structural elements, in both coding and non-coding regions, that are important for viral replication. These elements are known or predicted to modulate key life cycle events, such as translation and genome replication, some involving conformational changes induced by long-range RNA-RNA interactions. One such element is SLVI, a stem-loop (SL) structure located towards the 5' end of the core protein-coding region. This element forms an alternative RNA-RNA interaction with complementary sequences in the 5' untranslated regions that are independently involved in the binding of the cellular microRNA 122 (miR122). The switch between 'open' and 'closed' structures involving SLVI has previously been proposed to modulate translation, with lower translation efficiency associated with the 'closed' conformation. In the current study, we have used selective 2'-hydroxyl acylation analysed by primer extension to validate this RNA-RNA interaction in the absence and presence of miR122. We show that the long-range association (LRA) only forms in the absence of miR122, or otherwise requires the blocking of miR122 binding combined with substantial disruption of SLVI. Using site-directed mutations introduced to promote open or closed conformations of the LRA we demonstrate no correlation between the conformation and the translation phenotype. In addition, we observed no influence on virus replication compared to unmodified genomes. The presence of SLVI is well-documented to suppress translation, but these studies demonstrate that this is not due to its contribution to the LRA. We conclude that, although there are roles for SLVI in translation, the LRA is not a riboswitch regulating the translation and replication phenotypes of the virus.