Spontaneous dimerization of the hepatitis C virus 3′X RNA

The 3'X RNA of the hepatitis C virus is a highly conserved, 98-nucleotide, non-coding sequence located at the 3' terminus of the viral genome. This essential riboregulatory element contains a 16-nucleotide sequence, known as the dimer linkage sequence (DLS), which has been shown in vitro to facilitate the dimerization of the first 55 nucleotides of 3'X (3'X55). Here, we employ a novel integrative approach based on quantitative single-molecule FRET and analytical size-exclusion HPLC to monitor the structural, energetic, and dynamic aspects of 3'X55 dimerization. At high RNA concentrations, we see that 3'X55 can adopt multiple dimeric species with different structural properties. The interconversion between these species is slow, requiring several hours to reach equilibrium. Finally, the concentration- and time dependence of dimerization are both well described by a simple four-state kinetic model, which may explain how this riboregulatory interaction governs critical RNA-dependent processes during viral replication.