Programmable antivirals targeting critical conserved viral RNA secondary structures from influenza A virus and SARS-CoV-2.

Influenza A virus's (IAV's) frequent genetic changes challenge vaccine strategies and engender resistance to current drugs. We sought to identify conserved and essential RNA secondary structures within IAV's genome that are predicted to have greater constraints on mutation in response to therapeutic targeting. We identified and genetically validated an RNA structure (packaging stem-loop 2 (PSL2)) that mediates in vitro packaging and in vivo disease and is conserved across all known IAV isolates. A PSL2-targeting locked nucleic acid (LNA), administered 3 d after, or 14 d before, a lethal IAV inoculum provided 100% survival in mice, led to the development of strong immunity to rechallenge with a tenfold lethal inoculum, evaded attempts to select for resistance and retained full potency against neuraminidase inhibitor-resistant virus. Use of an analogous approach to target SARS-CoV-2, prophylactic administration of LNAs specific for highly conserved RNA structures in the viral genome, protected hamsters from efficient transmission of the SARS-CoV-2 USA_WA1/2020 variant. These findings highlight the potential applicability of this approach to any virus of interest via a process we term 'programmable antivirals', with implications for antiviral prophylaxis and post-exposure therapy.

Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome.

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.

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

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 nt 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, frameshifting stimulation element, and 3' UTR. Lastly, 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 accessible by hybridization in primer-based diagnostic strategies.