Shapify: Pathways to SARS-CoV-2 Frameshifting Pseudoknot

Abstract

Background: Multiple viruses including HIV, MERS-CoV (coronavirus responsible for Middle East Respiratory Syndrome, MERS), SARS-CoV (coronavirus responsible for SARS) and SARS-CoV-2 (coronavirus responsible for COVID-19) use a mechanism known as -1 programmed ribosomal frameshifting (-1 PRF) to successfully replicate. SARS-CoV-2 possesses a unique RNA pseudoknotted structure that stimulates -1 PRF. Recent experiments identified small molecules as antiviral agents that can bind to the pseudoknot and disrupt its stimulation of -1 PRF. Targeting -1 PRF in SARS-CoV-2 to impair viral replication can improve patients' prognoses.Crucial to developing these successful therapies is modeling the structure of the SARS-CoV-2 -1 PRF pseudoknot.Our goal is to expand knowledge of possible pseudoknot conformations. Results: Following a structural alignment approach, we identify similarities in -1 PRF pseudoknots of SARS-CoV-2, SARS-CoV, and MERS-CoV. We introduce Shapify, a novel algorithm that given an RNA sequence incorporates structural reactivity (SHAPE) data and partial structure information to output an RNA secondary structure prediction within a biologically sound hierarchical folding approach. Shapify helps us to better understand non-native SARS-CoV-2 -1 PRF pseudoknot conformations that are relevant to structure function and may correlate with -1 PRF efficiency. We provide in-depth analysis by investigating the structural landscape for the SARS-CoV-2 -1 PRF pseudoknot, including reference and mutated sequences. To better understand the impact of mutations, we provide insight on SARS-CoV-2 -1 PRF pseudoknot sequence mutations and their effect on the resulting structure. Conclusion: We identify the consensus structure for SARS-CoV, SARS-CoV-2, and MERS-CoV -1 PRF pseudoknots; this similarity in functional RNA structures aids treatment preparation for existing and emergent viruses. Shapify predictions are guided both by SHAPE data and partial structure information. Applied to the SARS-CoV-2 -1 PRF pseudoknot, Shapify unveiled previously unknown pathways from initial stems to pseudoknotted secondary structures. Where SHAPE data is unavailable we provide predictions for noteworthy SARS-CoV-2 -1 PRF mutated pseudoknot sequences. By contextualizing our work with available experimental data, our structure predictions motivate future RNA structure-function research and can aid 3-D modeling of pseudoknots.

To Knot or Not to Knot: Multiple Conformations of the SARS-CoV-2 Frameshifting RNA Element

“…Trinity et al 23 obtain a minor 3_8 and a 3_3 pseudoknot (Figure S13), but the corresponding 2D structures are different from those we have described. Recall that each graph topology corresponds to multiple 2D structures.…”

“…Recall that each graph topology corresponds to multiple 2D structures. In their 3_8 RNA, 23 base pairs in Stems 2 and 3 are the same as in our 3_6 in Figure 1, but the original Stem 1 is replaced by a pseudoknot that binds the 5′ end and the Stem 3 loop. In their 3_3, 23 Stem 3 loop and the 3′ end base pair to form the pseudoknot, instead of the 5′ end of the 77 nt FSE and Stem 1 loop.…”

“…Our MSA indicates that this AS1 may be Sarbecovirus-specific, and its covariation evidence is weaker than Stem 1 (Figure 2). Besides pseudoknots 3_6 and 3_3, three-way junction 3_5, and stem-loop 2_2, alternatives may include two 3_8 pseudoknots, 23,28 a different 3_3 pseudoknot (with Stem 2 formed by Stem 3 loop and 3′ end), 23 and a 2-stem 2_1 24,25 (Table 2 and Figure S13). Our work clearly shows that formation of Stem 1 and Alternative Stem 1 are mutually exclusive.…”