Mechanistic insights into the 2-step dimerization process FO the hepatitis C virus RNA genome.

Abstract

The 3ʹX RNA of the Hepatitis C Virus (HCV) is a highly conserved, 98-nt sequence located at the 3ʹ terminus of the HCV genome. This region of the genome can engage in several viral RNA-RNA binding interactions that have previously been shown to be critical for regulating translation, replication, and viral particle assembly. Within the 3ʹX RNA there is a stretch of 16 nucleotides that is responsible for two different RNA-RNA binding interactions: one heterotypic and the other homotypic. Notably, the nucleotide sequence for the heterotypic interaction is fully nested within the homotypic interaction and therefore formation of one interaction precludes formation of the other. Although these two interactions have important regulatory roles, it is not known what determines which interaction is formed at any given time and how they might interconvert. Using single-molecule FRET (smFRET) and size exclusion HPLC (SE-HPLC) techniques to monitor the conformational status of the 3ʹX RNA under a variety of conditions, we are beginning to resolve these unanswered questions. First, in the absence of either binding partner, we observe two distinct conformations of monomeric 3ʹX with their relative abundance dictated by either solution conditions (e.g., [Mg2+]) or nucleotide deletions. Additionally, we a observe large conformational change in the 3ʹX RNA when exposed to its homotypic binding partner, resulting in the formation of a homodimeric extended duplex of the 3ʹX RNA. These findings support an emerging hypothesis that the conformation changes associated with the 3ʹX RNA function as riboregulatory switches that halt protein and RNA synthesis, ultimately giving way for virus particles to begin to assemble.