The coronavirus causing the COVID-19 pandemic, SARS-CoV-2, uses −1 programmed ribosomal frameshifting (−1 PRF) to control the relative expression of viral proteins. As modulating −1 PRF can inhibit viral replication, the RNA pseudoknot stimulating −1 PRF may be a fruitful target for therapeutics treating COVID-19. We modeled the unusual 3-stem structure of the stimulatory pseudoknot of SARS-CoV-2 computationally, using multiple blind structural prediction tools followed by μs-long molecular dynamics simulations. The results were compared for consistency with nuclease-protection assays and single-molecule force spectroscopy measurements of the SARS-CoV-1 pseudoknot, to determine the most likely conformations. We found several possible conformations for the SARS-CoV-2 pseudoknot, all having an extended stem 3 but with different packing of stems 1 and 2. Several conformations featured rarely-seen threading of a single strand through junctions formed between two helices. These structural models may help interpret future experiments and support efforts to discover ligands inhibiting −1 PRF in SARS-CoV-2.
The coronavirus causing the COVID-19 pandemic, SARS-CoV-2, uses −1 programmed ribosomal frameshifting (−1 PRF) to control the relative expression of viral proteins. As modulating −1 PRF can inhibit viral replication, the RNA pseudoknot stimulating −1 PRF may be a fruitful target for therapeutics treating COVID-19. We modeled the unusual 3stem structure of the stimulatory pseudoknot of SARS-CoV-2 computationally, using multiple blind structural prediction tools followed by μs-long molecular dynamics simulations. The results were compared for consistency with nuclease-protection assays and single-molecule force spectroscopy measurements of the SARS-CoV-1 pseudoknot, to determine the most likely conformations. We found several possible conformations for the SARS-CoV-2 pseudoknot, all having an extended stem 3 but with different packing of stems 1 and 2. Several conformations featured rarely-seen threading of a single strand through the junction formed between two helices. These structural models may help interpret future experiments and support efforts to discover ligands inhibiting −1 PRF in SARS-CoV-2.
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.
SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, belongs to the Coronaviridae family of viruses. Members of this family contain highly conserved structural elements within their 5' and 3' untranslated regions (UTRs), which are believed to play roles in genome replication and circularization. One structure of interest in the 3'-UTR is a highly conserved 41-nucleotide stem-loop II-like motif (s2m), which is found not only within coronaviruses, but also within Astroviridae, Calciviridae, and Picornaviridae families. While the specific function remains unresolved, it has been suggested to play roles in viral replication, hijacking of host protein synthesis, and RNA interference pathways. Here, we demonstrate the formation of a homodimeric kissing complex by the SARS-CoV-2 s2m element, which is converted to a stable, extended duplex by the viral nucleocapsid (N) protein. Subsequently, we establish a potential role of host cellular microRNA-1307-3p hijacking by the s2m element, a function which is altered by s2m dimerization. The purported roles of this microRNA include translational regulation of various interleukins and interleukin receptors, including those found elevated in severe COVID-19 patients. Furthermore, we have identified a second region in the 3'-UTR of SARS-CoV-2 that binds another host miRNA involved in interleukin expression. Our data indicates that the SARS-CoV-2 virus may act as a ''microRNA sponge'', sequestering host cellular microRNAs and preventing them from performing their canonical functions. The molecular mechanisms put forth in this study could act as a guide for future therapeutic drug development studies in order to alleviate the severe symptoms associated with COVID-19.
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