Abstract
The 3' end of the hepatitis C virus genome is terminated by a highly conserved, 98 nt sequence called 3'X. This untranslated structural element is thought to regulate several essential RNA-dependent processes associated with infection. 3'X has two proposed conformations comprised of either three or two stem-loop structures that result from the different base-pairing interactions within the first 55 nt. Here, we used single-molecule Förster resonance energy transfer spectroscopy to monitor the conformational status of fluorescently labeled constructs that isolate this region of the RNA (3'X55). We observed that 3'X55 can adopt both proposed conformations and the relative abundance of them can be modulated by either solution conditions or nucleotide deletions. Furthermore, interconversion between the two conformations takes place over the course of several hours. The simultaneous existence of two slowly interconverting conformations may help prime individual copies of the viral genome for either viral protein or RNA synthesis, thereby minimizing conflicts between these two competing processes.