Uncovering the Biochemical Mechanisms of Riboregulatory Interaction Involving the Hepatitis C Virus 3'X RNA

PROJECT SUMMARY The hepatitis C virus (HCV) is a nanoscopic lipid-enveloped biological entity. Despite the existence of highly effective treatment plans, this pathogenic particle remains responsible for thousands of deaths each year in the United States. At the core of this particle lies a genetic message comprised of a single RNA molecule, the vast

majority of which encodes for a roughly 3000 amino acid-long polyprotein. However, there are 98 nucleotides at the very end of the single-stranded HCV genome that do not encode proteins and yet are essential for the virus. These nucleotides make up the so-called 3ʹX RNA, which is a structured and highly conserved RNA element

that has been implicated in the regulation of several different RNA-dependent viral processes like replication, translation, and particle assembly. Many research groups have proposed that this riboregulatory behavior arises from this RNA's ability to form mutually exclusive interactions with other highly conserved RNA elements within

the HCV genome. Unfortunately, the fundamental biochemical principles governing these RNA-RNA interactions have not yet been established, which greatly limits our ability to predict which of these mutually exclusive interactions will be formed under specific conditions and ultimately how the formation and disruption of them

regulate various viral processes. This research project will begin to address these unknowns by determining the major structural, energetic, and kinetic principles that govern these riboregulatory interactions involving the 3ʹX RNA. These fundamental insights will be obtained by studying fluorescently labeled HCV RNAs at the single-

molecule level using FRET spectroscopy and microscopy with additional support from cryo-electron microscopy as well as more conventional electrophoretic and chromatographic approaches. Our findings will allow us to construct a quantitative physical model that highlights the biochemical function of this highly conserved non-

coding viral RNA and its various interaction partners. Not only will this knowledge enhance our understanding of HCV and the diseases it causes, but because these regulatory RNA-RNA interactions often appear in other non- viral RNAs, it will also positively impact several other areas of general RNA biology.