Functional implications of structural heterogeneity in a viral RNA translation initiation element - PROJECT SUMMARY
RNA viruses are an emerging health threat, evolving rapidly to proliferate and spread. Within these viruses are
structured RNA elements that facilitate infection and virus survival. Internal Ribosome Entry Sites (IRESs) are a
type of RNA structure found within RNA viruses that facilitate infection by enabling initiating translation on the
viral genome when cellular translation is repressed during infection. The Hepatitis C Virus (HCV) contains an
IRES that has been extensively studied, demonstrating a mechanism of initiation intimately linked to RNA
architecture. The HCV IRES consists of a large, multi-domain structure that interacts directly with the small
ribosomal subunit (40S) and eukaryotic initiation factor 3 (eIF3) to form an initiation complex. In tandem with
mechanistic and structural studies of the HCV IRES, a handful of other viruses with a similar structure– termed
HCV-like IRESs – have been sporadically identified through sequence homology. Despite demonstrating notable
structural diversity, HCV-like IRESs are inferred to share the same mechanism. I hypothesize that there are
distinct structural subgroups within the HCV class of IRESs and between these subgroups there are
differences in their initiation complex components and intermolecular interactions. In my first Aim, I will
define the structural diversity of HCV-like IRESs as a class using computational and biochemical methods. I
have performed a structure alignment-based search to mine the NCBI virus database, revealing 178 unique
putative IRESs. I will use computational methods to structurally characterize discrete regions of these putative
IRESs that differ between subgroups. Predicted IRES architecture and function will be validated biochemically
to link structural heterogeneity with translation initiation efficiency. For Aim 2, I will uncover the mechanistic basis
underlying the structural and functional differences among HCV-like IRES by investigating initiation complex
formation. I will determine both the composition of these IRES initiation complexes and the affinity of the IRES
for each component within the complex. Select IRES initiation complexes will be visualized using cryo-electron
microscopy, potentially revealing novel intermolecular interactions. The work outlined in this proposal will
enhance understanding of the relationship between HCV-like IRES structure and function. Further, this research
will provide insight into mechanisms of eukaryotic initiation that could be leveraged for use in vaccines and
therapeutics to combat RNA viruses.
