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The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the COVID-19 pandemic has highlighted the need for rapid characterization of the viral mechanisms responsible for cellular pathogenesis. Viral untranslated regions (UTRs) represent conserved genomic elements that contribute to such mechanisms, and that are considered to be targets for therapeutic intervention. Details of the structures of most coronavirus (CoV) UTRs are not available, however. Experimental approaches are needed to allow for the facile generation of high-quality viral RNA tertiary structural models, which can facilitate comparative mechanistic and drug discovery efforts. By integrating experimental and computational techniques, we herein report the efficient characterization of conserved RNA structures within the 5’UTR of the human coronavirus OC43 (HCoV-OC43) genome, a lab tractable model coronavirus. Evidence is provided that the 5’UTR folds into a secondary structure with well-defined stem loops (SLs) as determined by chemical probing and direct detection of hydrogen bonds by NMR spectroscopy. We combine experimental base-pair restraints with global structural information from SAXS to generate a 3D model that reveals that SL1-4 adopts a topologically constrained structure wherein stem loops 3 and 4 co-axially stack. Co-axial stacking is mediated by short linker nucleotides and allows stem loops 1-2 to sample different co-joint orientations by pivoting about the SL3,4 helical axis. To evaluate the functional relevance of the SL3,4 co-axial helix, luciferase reporter constructs harboring the HCoV-OC43 5’UTR were engineered to contain mutations designed to abrogate co-axial stacking. The results reveal that the SL3,4 helix intrinsically represses translation efficiency since the destabilizing mutations correlate with increased luciferase expression relative to wild-type without affecting reporter mRNA levels. We also show that the nucleocapsid protein engages differentially with the TRS-L depending on its structural context. SAXS measurements were again combined with molecular dynamics in order to develop a data-driven model for the interaction between the nucleocapsid protein and SLs 1-4 of the HCoV-OC43 UTR suggesting a complex and dynamic interaction which further constrains the SL 1-4 locus. Drawing on these observations, interactions between the nucleocapsid protein and the 5’UTR were investigated using biophysical characterization methods in order to model the binding interface between protein and RNA. In sum, the work presented herein describes an efficient approach to discover a functionally relevant tertiary interaction within the HCoV-OC43 5’UTR which provides a potential druggable target for the suppression of coronavirus pathogenesis. |
