Wednesday, April 30, 2025
4/30/2025
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.
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