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...

T he current pandemic caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2),continues to spread, and as of April 1, 2020, more than 920,000 cases have been reported worldwide. 1 Although the respiratory complications of SARS-CoV-2 have been described, the impact on the gastrointestinal and hepatic systems remains unknown, with conflicting levels noted in preliminary cases from China and Singapore. [2][3][4] We aim to characterize the gastrointestinal manifestations in patients with SARS-CoV-2 at our institution.

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5′ end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.

Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 Å, presents a topologically complex fold in which the 5′ end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.

Development of cancer therapeutics requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Three-dimensional (3-D) in vitro models have therefore been investigated for drug screening. In this study, we have developed a novel in vitro model in which multicellular aggregates, or spheroids, were incorporated into 3-D porous scaffolds. Drug resistance assays showed that spheroid-seeded scaffolds have much higher drug resistance than monolayer cultures, spheroids on flat substrates, or scaffolds seeded with dispersed cells. Furthermore, spheroid-seeded scaffolds demonstrated higher lactate production leading to acidosis, and higher expression of angiogenic factors. These data suggest that the spheroid-seeded 3-D scaffolds might serve as a useful in vitro system for screening cancer therapeutics. (Cancer Sci 2010; 101: 2637-2643