ABSTRACT
Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5' end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.
Subject(s)
COVID-19/prevention & control , Cryoelectron Microscopy/methods , Frameshift Mutation/genetics , Oligonucleotides, Antisense/genetics , RNA, Viral/genetics , Response Elements/genetics , SARS-CoV-2/genetics , A549 Cells , Animals , Base Sequence , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Genome, Viral/genetics , Humans , Models, Molecular , Nucleic Acid Conformation , Oligonucleotides, Antisense/pharmacology , RNA, Viral/chemistry , RNA, Viral/ultrastructure , SARS-CoV-2/physiology , SARS-CoV-2/ultrastructure , Vero Cells , Virus Replication/drug effects , Virus Replication/geneticsABSTRACT
The RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target. To understand how it responds to mechanical tension applied by ribosomes, thought to play a key role during frameshifting, we probe its structural dynamics using optical tweezers. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. The pseudoknotted conformers have distinct topologies, one threading the 5' end through a 3-helix junction to create a knot-like fold, the other with unthreaded 5' end, consistent with structures observed via cryo-EM and simulations. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2; Mg2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer. These results resolve the SARS-CoV-2 frameshift signal folding mechanism and highlight its conformational heterogeneity, with important implications for structure-based drug-discovery efforts.