Coronaviruses (CoVs) use -1 programmed ribosomal frameshifting stimulated by RNA pseudoknots in the viral genome to control expression of enzymes essential for replication, making CoV pseudoknots a promising target for anti-coronaviral drugs. Bats represent one of the largest reservoirs of CoVs and are the ultimate source of most CoVs infecting humans, including those causing SARS, MERS, and COVID-19. However, the structures of bat-CoV frameshift-stimulatory pseudoknots remain largely unexplored. Here we use a combination of blind structure prediction followed by all-atom molecular dynamics simulations to model the structures of eight pseudoknots that, together with the SARS-CoV-2 pseudoknot, are representative of the range of pseudoknot sequences in bat CoVs. We find that they all share some key qualitative features with the pseudoknot from SARS-CoV-2, notably the presence of conformers with two distinct fold topologies differing in whether or not the 5 ' end of the RNA is threaded through a junction, and similar conformations for stem 1. However, they differed in the number of helices present, with half sharing the 3-helix architecture of the SARS-CoV-2 pseudoknot but two containing 4 helices and two others only 2. These structure models should be helpful for future work studying bat-CoV pseudoknots as potential therapeutic targets. Author summaryStructures in coronavirus (CoV) genomes called pseudoknots control expression of viral proteins essential for replication, making them attractive targets for broad-spectrum anti-CoV drugs. However, the 3D structures of most CoV pseudoknots are unknown. Here we computationally model the structures of a set of pseudoknots that are representative of the range of sequences found in bat-CoV pseudoknots, motivated by the fact that bat CoVs are the ultimate source of most human CoV diseases. We find these representative pseudoknots all share some crucial structural features, including very similar configurations of the central helix in the pseudoknot and an unusual topology in which one end of the RNA threads through a ring formed in the structure. The commonality of these features across bat-CoV pseudoknots supports the notion that drugs may be able to bind many different CoV pseudoknots, and the rarity of the fold topology suggests such binding should be highly specific to CoVs, implying pseudoknots are well-suited as targets for broad-spectrum anti-CoV drugs. This work provides new insights into CoV pseudoknot structures that may help future efforts targeting pseudoknots therapeutically.
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