Shapify: Paths to SARS-CoV-2 frameshifting pseudoknot

Author summaryIdentifying inter-viral structural similarity in frameshifting pseudoknots is valuable for treatment development as existing viruses mutate or novel diseases emerge. Computational methods for RNA secondary structure prediction remain critical tools for understanding functional structures, and advancing knowledge for the development of possible treatment options. However, current understanding of the full landscape of potential structures for any given RNA is limited by the cost and complexity of experimental methods and intractability of comprehensive algorithmic approaches.We followed a structural alignment approach to identify the consensus structure for SARS-CoV and MERS-CoV -1 PRF pseudoknots, with SARS-CoV-2 as reference. We developed Shapify for improved prediction of possibly pseudoknotted RNA secondary structures guided by SHAPE reactivity data. Then, we shed light on the structure formation of the SARS-CoV-2 and MERS-CoV frameshifting pseudoknots by analyzing possible structural conformations. Previous structural predictions obtained via different methods all have significant differences. Our results demonstrate innate resiliency via converging paths of the SARS-CoV-2 virus in achieving the native -1 PRF stimulating pseudoknot. Fully accounting for pan-coronaviral structural conformations, which include transient and suboptimal structures, is vital for comprehending viral function. Multiple coronaviruses including MERS-CoV causing Middle East Respiratory Syndrome, SARS-CoV causing SARS, and SARS-CoV-2 causing COVID-19, use a mechanism known as -1 programmed ribosomal frameshifting (-1 PRF) to replicate. SARS-CoV-2 possesses a unique RNA pseudoknotted structure that stimulates -1 PRF. Targeting -1 PRF in SARS-CoV-2 to impair viral replication can improve patients' prognoses. Crucial to developing these therapies is understanding the structure of the SARS-CoV-2 -1 PRF pseudoknot. Our goal is to expand knowledge of -1 PRF structural conformations. Following a structural alignment approach, we identify similarities in -1 PRF pseudoknots of SARS-CoV-2, SARS-CoV, and MERS-CoV. We provide in-depth analysis of the SARS-CoV-2 and MERS-CoV -1 PRF pseudoknots, including reference and noteworthy mutated sequences. To better understand the impact of mutations, we provide insight on -1 PRF pseudoknot sequence mutations and their effect on resulting structures. 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 enhances our understanding of SARS-CoV-2 -1 PRF pseudoknot conformations by providing energetically favourable predictions that are relevant to structure-function and may correlate with -1 PRF efficiency. Applied to the SARS-CoV-2 -1 PRF pseudoknot, Shapify unveils previously unknown paths from initial stems to pseudoknotted structures. 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.