Structural and mechanistic studies of cap-independent genome translation in (+)-strand RNA viruses

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Project Summary Our main goal is to understand how internal ribosome entry sites (IRESs) and 3' cap-independent translation enhancers (3'CITEs) promote cap-independent translation of genomes in (+)-strand RNA viruses. Despite highly diverse sequences and predicted secondary structures, IRESs and 3ʹCITEs from evolutionarily distant viruses recruit the same components from the host to initiate genome translation. Structural information for viral IRESs that bind directly to the ribosome is limited and understanding of 3-dimensional (3D) structures and interactions of IRESs and 3'CITEs that promote translation by other mechanisms is largely unknown. We will use X-ray crystallography to determine the high-resolution crystal structures of these RNAs, focusing on the type I and type II picornaviral IRESs and tombusvirus 3'CITEs due to their unique mechanisms of recruiting translation initiation factors and the ribosomal subunits through a multistep, dynamic assembly process using modular RNA domains. Our strategy employs Fab fragments as chaperones to crystallize and determine structures of RNAs and RNPs, an extension of a technology that PI helped develop as a postdoc. Since moving to UMBC, we have obtained crystals of coxsackievirus IRES domain V (an example of type I IRES) in a complex with Fab BL3-6 that diffracted to 3.36 Å resolution (the first 3D structural information for type I IRESs). A partial structure of the dV contains a 3-way junction analogous to that observed in cardiovirus J-K and hepatitis A virus dV structures (PI's previous work). Optimization of crystallization conditions to obtain high-resolution diffraction data, analysis of SAXS data to access in-solution structural information, and purification of the human eIF4G HEAT-1 domain for binding studies are underway. Recently, we solved the 2.9 Å resolution crystal structure of a T-shaped domain of saguaro cactus virus 3ꞌCITE in a complex with Fab BL3-6 and characterized its binding with human eIF4E. Many viruses within the tombusviridae family contain 3ꞌCITEs with similar domains, suggesting that these RNAs adopt a shared topology to mimic mRNA 5ꞌ-cap for binding eIF4E. We are thus poised to determine the crystal structures of different kinds of viral IRESs, 3'CITEs, and some cellular IRESs. The Fab approach has successfully solved the crystal structures of several RNAs, but it has not been demonstrated for RNP complexes; the second goal is to integrate this Fab-assisted technology to crystallize and determine the structures of RNP complexes. The third goal is to create anti-RNA single-chain variable fragments (scFvs) as unique probes for RNA visualization to facilitate the localization, tracking, and quantification of viral RNAs in host cells. We have obtained promising preliminary data in this direction, including the development of three anti-RNA scFvs and their scFv-GFP fusions based on the existing anti-RNA Fabs. The scFvs with and without GFP tags bind the cognate RNA targets with affinities similar to their parent Fabs. When taken together, the proposed studies will provide deeper insights into the mechanisms of cap-independent translation initiation in (+)-sense RNA viruses and unlock opportunities for developing RNA-targeted therapeutics against these pathogens that cause human, animal, and plant diseases.