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

Abstract

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.

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

“…Incomplete inactivation of coronavirus frameshifting and replication in these and prior studies motivated structural analysis to better understand the FSE. Despite prior expectations of structural heterogeneity and propensity for multimerization 4,45,52 , and despite having a size slightly under that of the previous smallest macromolecule imaged by cryo-EM ( We note two caveats regarding our cryo-EM analysis. First, the 6.9 Å resolution of the cryo-EM map is not sufficient to directly resolve base interactions, much less atom positions.…”

“…The urgency of the COVID-19 pandemic, recent advances in targeting RNA 3D structures with ASOs and small molecules, and the identification of the FSE as a potentially well-defined RNA 3D structure in the SARS-CoV-2 genome has generated significant interest in understanding and targeting SARS-CoV-2 ribosomal frameshifting 3,23,27,28,44,45,50,51 . Here, we confirmed the ability of ASOs to invade the SARS-CoV-2 FSE structure and to reduce frameshifting efficiencies in cellfree assays, and we explored the potential for these ASOs to reduce viral replication in cells at submicromolar concentrations.…”

“…There is accumulating evidence that, in the full SARS-CoV-2 genome context, the FSE forms alternative structures involving genomic segments upstream of the element (Extended Data Fig. 13 and refs 27,44,45,50 ). As the human ribosome approaches the FSE, it must unfold these upstream structures through its helicase activity, and the FSE will refold.…”

“…The 5′ end of the FSE is connected to the pseudoknot by a linker that passes through this ring. This complex topology was predicted as a possible FSE fold in two recent, independent 3D computer modeling studies that include an extended 5′ end 44,45 . We emphasize that while the fold of the 5′ end appears poorly resolved and thus may have multiple conformations, the connection point of the 5′ end to the rest of the structure and its threading through the Supporting the general relevance of 5′-end ring-threading, the length of Stem 1 is ~10 base pairs or larger in all proposed coronavirus FSE elements 22 , and de novo modeling of FSEs from bovine coronavirus, murine hepatitis virus, human coronaviruses OC43 and HKU1, SARS-CoV-1, and MERS all give 5′ ring-threaded models (Extended Data Figure 9).…”

“…It is the smallest macromolecule (28 kDa) so far resolved by cryo-EM single particle analysis at subnanometer resolution. We therefore sought further independent validation of the map and the Ribosolve model, particularly to test our inference that Stem 3 comprises a ′leg′ of the λ shape, perpendicular to the Stem 1-Stem 2 pseudoknot 44,45 . We rationally designed a variant of the SARS-CoV-2 FSE termed FSE-ATP-TTR3, which contains an insertion of a clothespin-like nanostructure whose visualization would test the assignment and orientation of Stem 3 in the FSE map 46 (Fig.…”