Modeling the structure of the frameshift-stimulatory pseudoknot in SARS-CoV-2 reveals multiple possible conformers

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.Author summary: The coronavirus that causes COVID-19 controls the production of key viral proteins through a process known as programmed ribosomal frameshifting. Frameshifting is triggered by a particular structure in the viral RNA, a pseudoknot, which is a promising drug target. Here we model the structure of this pseudoknot through atomistic molecular dynamics simulations. Surprisingly, we find that the pseudoknot can take on distinct fold topologies, two of which involve unusual threading of a single strand of RNA through helical junctions, something not seen before in frameshifting pseudoknots. All of the folds are generally consistent with previous experimental studies of the closely-related SARS coronavirus pseudoknot. These results should assist in the analysis and interpretation of future experimental studies of the pseudoknot structure, and support structure-based drug-discovery efforts.