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.
