RESUMEN
The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.
Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Exorribonucleasas/metabolismo , Genoma Viral/genética , Inestabilidad Genómica , SARS-CoV-2/enzimología , SARS-CoV-2/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Exorribonucleasas/antagonistas & inhibidores , Genoma Viral/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/genética , Inhibidores de Integrasa VIH/farmacología , Isoindoles/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Compuestos de Organoselenio/farmacología , ARN Viral/biosíntesis , ARN Viral/genética , Raltegravir Potásico/farmacología , SARS-CoV-2/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.