RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses: a first look

Abstract

As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Here we present a first look at RNA sequence conservation and structural propensities in the SARS-CoV-2 genome. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nucleotides as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences subsequently reported from the COVID-19 outbreak, and we present a curated list of 30 'SARS-related-conserved' regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 'SARS-CoV-2-conserved-structured' regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the extended 5´ UTR, frame-shifting element, and 3´ UTR. Last, we predict regions of the SARS-CoV-2 viral genome that have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 'SARS-CoV-2-conserved-unstructured' genomic regions may be most easily targeted in primer-based diagnostic and oligonucleotidebased therapeutic strategies.Cold Spring Harbor Laboratory Press on June 9, 2020 -Published by rnajournal.cshlp.org Downloaded from , Connelly, et al. 2016, Spurgers, et al. 2008).Conserved structured RNA regions have already been shown to play critical functional roles in the life cycles of coronaviruses. Most coronavirus 5´ UTR's harbor at least four stem loops, with many showing heightened sequence conservation across betacoronaviruses, and various stems demonstrating functional roles in viral replication (Yang and Leibowitz 2015). Furthermore, RNA secondary structure in the 5´ UTR exposes a critical sequence motif, the transcriptional regulatory sequence (TRS), that forms long-range RNA interactions necessary for facilitating the discontinuous transcription characteristic to coronaviruses (van den Born, et al. 2005). Beyond the 5´ UTR, the frame-shifting element (FSE) in the first protein-coding ORF (ORF1ab) includes a pseudoknot structure that is necessary for the production of ORF1a and ORF1b from two Cold Spring Harbor Laboratory Press on June 9, 2020 -Published by rnajournal.cshlp.org Downloaded from Results RNA sequence conservation in SARS-related betacoronaviruses and SARS-CoV-2 Cold Spring Harbor Laboratory Press on June 9, 2020 -Published by rnajournal.cshlp.org Downloaded from 1. The first multiple sequence alignment (SARSr-MSA-1) was computed by aligning sequences curated by Ceraolo and Giorgi (Ceraolo and Giorgi 2...

The global and local distribution of RNA structure throughout the SARS-CoV-2 genome

“…Consistent with reports on other coronaviruses, the SARS-CoV-2 genome contains highly conserved RNA structural elements that likely play pivotal roles in viral replication, including several structures in the UTRs and a ribosomal frameshifting element (Rangan et al 2020).…”

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

“…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.…”

Comprehensive in-vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms

“…3C, median Nuc(13476-13503) = 1.9x10 -4 ; global median = 0.022). Importantly, the region reported to contain the SL2 stem (Rangan et al, 2020, Plant et al, 2005) is predicted as single-stranded in our consensus structure, and consequently does not include SL2 (Fig 3A; region indicated by dotted black line). Rather, the dominant structure predicted for the PRF includes a different stem, SL3, that includes the downstream pseudoknot arm (Fig 3A; labeled SL3).…”