Abstract
The programmed −1 ribosomal frameshifting element (PFSE) of SARS-CoV-2 is a well conserved structured RNA found in all coronaviruses’ genomes. By adopting a pseudoknot structure in the presence of the ribosome, the PFSE promotes a ribosomal frameshifting event near the stop codon of the first open reading frame Orf1a during translation of the polyprotein pp1a. Frameshifting results in continuation of pp1a via a new open reading frame, Orf1b, that produces the longer pp1ab polyprotein. Polyproteins pp1a and pp1ab produce nonstructural proteins NSPs 1–10 and NSPs 1–16, respectively, which contribute vital functions during the viral life cycle and must be present in the proper stoichiometry. Both drugs and sequence alterations that affect the stability of the −1 programmed ribosomal frameshifting element disrupt the stoichiometry of the NSPs produced, which compromise viral replication. For this reason, the −1 programmed frameshifting element is considered a promising drug target. Using chaperone assisted RNA crystallography, we successfully crystallized and solved the three-dimensional structure of the PFSE. We observe a three-stem H-type pseudoknot structure with the three stems stacked in a vertical orientation stabilized by two triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions. This structure provides a new conformation of PFSE distinct from the bent conformations inferred from midresolution cryo-EM models and provides a high-resolution framework for mechanistic investigations and structure-based drug design.
Highlights
The historic and deadly COVID-19 pandemic is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV-2)
It is possible that our Fab-hairpin crystallization module and crystal packing forces facilitated formation of the linear conformation we observe in this crystal structure
The biological relevance of the linear conformation and associated base triples observed in this high-resolution structure await further investigation either in the context of frameshifting or in another stage of the viral lifecycle
Summary
The historic and deadly COVID-19 pandemic is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV-2). A lack of high-resolution, three-dimensional structural information about structured regions of the genome make development of drugs to target them difficult. Computational modeling and structural probing techniques are able to identify structured regions within RNAs and can suggest whether an RNA element might contain a pocket sufficient for ligand binding, but these estimations often lack certainty about the chemical arrangement of binding pockets.[1,2] By contrast, broad screens of RNA binding chemicals do not require high resolution structural information as a starting point and can yield lead molecules, but these chemicals are rarely drug-like due to their toxicity, lack of cell permeability, or lack of bioavailability.[1] Experimentally derived structures of viral RNA elements can provide another route to drug discovery. Existing drugs can be screened against experimentally determined structural models using structural dynamics simulations to identify potential binders.[3]
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