RESUMO
SARS-CoV-2 is a betacoronavirus that causes COVID-19, a global pandemic that has resulted in many infections, deaths, and socio-economic challenges. The virus has a large positive-sense, single-stranded RNA genome of â¼30 kb, which produces subgenomic RNAs (sgRNAs) through discontinuous transcription. The most abundant sgRNA is sgRNA N, which encodes the nucleocapsid (N) protein. In this study, we probed the secondary structure of sgRNA N and a shorter model without a 3' UTR in vitro, using the SHAPE (selective 2'-hydroxyl acylation analyzed by a primer extension) method and chemical mapping with dimethyl sulfate and 1-cyclohexyl-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate. We revealed the secondary structure of sgRNA N and its shorter variant for the first time and compared them with the genomic RNA N structure. Based on the structural information, we designed gapmers, siRNAs and antisense oligonucleotides (ASOs) to target the N protein coding region of sgRNA N. We also generated eukaryotic expression vectors containing the complete sequence of sgRNA N and used them to screen for new SARS-CoV-2 gene N expression inhibitors. Our study provides novel insights into the structure and function of sgRNA N and potential therapeutic tools against SARS-CoV-2.
Assuntos
Conformação de Ácido Nucleico , RNA Viral , SARS-CoV-2 , Replicação Viral , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Replicação Viral/efeitos dos fármacos , RNA Viral/genética , Humanos , Antivirais/farmacologia , Antivirais/química , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/antagonistas & inibidores , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/química , Ésteres do Ácido Sulfúrico/farmacologia , Ésteres do Ácido Sulfúrico/química , COVID-19/virologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/química , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/química , Genoma Viral , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/químicaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic, whereas the influenza A virus (IAV) causes seasonal epidemics and occasional pandemics. Both viruses lead to widespread infection and death. SARS-CoV-2 and the influenza virus are RNA viruses. The SARS-CoV-2 genome is an approximately 30 kb, positive sense, 5' capped single-stranded RNA molecule. The influenza A virus genome possesses eight single-stranded negative-sense segments. The RNA secondary structure in the untranslated and coding regions is crucial in the viral replication cycle. The secondary structure within the RNA of SARS-CoV-2 and the influenza virus has been intensively studied. Because the whole of the SARS-CoV-2 and influenza virus replication cycles are dependent on RNA with no DNA intermediate, the RNA is a natural and promising target for the development of inhibitors. There are a lot of RNA-targeting strategies for regulating pathogenic RNA, such as small interfering RNA for RNA interference, antisense oligonucleotides, catalytic nucleic acids, and small molecules. In this review, we summarized the knowledge about the inhibition of SARS-CoV-2 and influenza A virus propagation by targeting their RNA secondary structure.
