Architecture of a divergent bipartite internal ribosome entry site from Giardiavirus

Project summary Translation initiation is the most regulated step in protein synthesis. In eukaryotes, canonical translation initiation begins with recognition of a cap structure on the mRNA 5’ end, and is a very complex process requiring many protein factors and intermediates. To favor translation of their own proteins, RNA viruses have evolved simpler ways to initiate protein synthesis. One such viral tool is an internal RNA entry site, or IRES, a structured RNA that drives noncanonical, cap-independent translation. Some IRES elements show great promise, either as new targets for therapies directed against pathogenic RNA viruses, or as tools for optimizing protein expression in mRNA-based vaccines. The three-dimensional IRES structures determined to date have come almost exclusively from just two of the many IRES classes. More structures of diverse IRES elements are needed to determine existing IRES mechanisms, and ultimately to lay the groundwork for the development of improved IRES tools for novel therapies. Despite their significant structural and mechanistic differences, almost all IRES elements share one notable feature: little if any of the IRES is located in the coding region. Remarkably, a IRES from Giardia lamblia virus (GLV) violates this paradigm. The GLV IRES is bipartite, with one canonical domain 5’ to the start codon, and a second large, highly structured domain located entirely in its coding region. The secondary and three-dimensional structures of the GLV IRES are unknown. We propose to determine the architecture of the GLV IRES bound to its host ribosome. This work will provide foundational knowledge about the role of an unexplored class of IRES domain in viral infection of a human pathogen, Giardia lamblia. Giardiasis is the most common parasitic human diarrheal disease worldwide, and an increasingly serious neglected disease. Our results will pave the way for long-term studies of this divergent IRES mechanism, and potentially reveal pathways that can be targeted in diverse viruses, helping the long-term goal of developing new therapies for one of the world’s leading intestinal diseases.