RESUMEN
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.
Asunto(s)
COVID-19/prevención & control , Microscopía por Crioelectrón/métodos , Mutación del Sistema de Lectura/genética , Oligonucleótidos Antisentido/genética , ARN Viral/genética , Elementos de Respuesta/genética , SARS-CoV-2/genética , Células A549 , Animales , Secuencia de Bases , COVID-19/virología , Línea Celular Tumoral , Chlorocebus aethiops , Genoma Viral/genética , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/farmacología , ARN Viral/química , ARN Viral/ultraestructura , SARS-CoV-2/fisiología , SARS-CoV-2/ultraestructura , Células Vero , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
The rapid spread of COVID-19 is motivating development of antivirals targeting conserved SARS-CoV-2 molecular machinery. The SARS-CoV-2 genome includes conserved RNA elements that offer potential small-molecule drug targets, but most of their 3D structures have not been experimentally characterized. Here, we provide a compilation of chemical mapping data from our and other labs, secondary structure models, and 3D model ensembles based on Rosetta's FARFAR2 algorithm for SARS-CoV-2 RNA regions including the individual stems SL1-8 in the extended 5' UTR; the reverse complement of the 5' UTR SL1-4; the frameshift stimulating element (FSE); and the extended pseudoknot, hypervariable region, and s2m of the 3' UTR. For eleven of these elements (the stems in SL1-8, reverse complement of SL1-4, FSE, s2m and 3' UTR pseudoknot), modeling convergence supports the accuracy of predicted low energy states; subsequent cryo-EM characterization of the FSE confirms modeling accuracy. To aid efforts to discover small molecule RNA binders guided by computational models, we provide a second set of similarly prepared models for RNA riboswitches that bind small molecules. Both datasets ('FARFAR2-SARS-CoV-2', https://github.com/DasLab/FARFAR2-SARS-CoV-2; and 'FARFAR2-Apo-Riboswitch', at https://github.com/DasLab/FARFAR2-Apo-Riboswitch') include up to 400 models for each RNA element, which may facilitate drug discovery approaches targeting dynamic ensembles of RNA molecules.
Asunto(s)
Consenso , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Viral/química , SARS-CoV-2/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Algoritmos , Aptámeros de Nucleótidos/genética , Secuencia de Bases , Sitios de Unión , Microscopía por Crioelectrón , Conjuntos de Datos como Asunto , Evaluación Preclínica de Medicamentos/métodos , Sistema de Lectura Ribosómico/genética , Genoma Viral/genética , Estabilidad del ARN , ARN Viral/genética , Reproducibilidad de los Resultados , Riboswitch/genética , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
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.