RESUMEN
The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS-CoV-2 and SARS-CoV share an otherwise non-conserved part of non-structural protein 3 (Nsp3), therefore named as "SARS-unique domain" (SUD). We previously found a yeast-2-hybrid screen interaction of the SARS-CoV SUD with human poly(A)-binding protein (PABP)-interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS-CoV SUD:Paip1 interaction by size-exclusion chromatography, split-yellow fluorescent protein, and co-immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS-CoV-2 and Paip1. The three-dimensional structure of the N-terminal domain of SARS-CoV SUD ("macrodomain II", Mac2) in complex with the middle domain of Paip1, determined by X-ray crystallography and small-angle X-ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC-SARS-CoV replicon-transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS-CoV and SARS-CoV-2.
Asunto(s)
Proteasas Similares a la Papaína de Coronavirus/metabolismo , Regulación Viral de la Expresión Génica , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2/fisiología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas , Cromatografía en Gel , Proteasas Similares a la Papaína de Coronavirus/química , Cristalografía por Rayos X , Genes Reporteros , Células HEK293 , Humanos , Inmunoprecipitación , Proteínas Luminiscentes , Modelos Moleculares , Factores de Iniciación de Péptidos/química , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas , ARN Viral/genética , Proteínas de Unión al ARN/química , ARN Polimerasa Dependiente del ARN/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Subunidades Ribosómicas/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , SARS-CoV-2/genética , Dispersión del Ángulo Pequeño , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/química , Difracción de Rayos XRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40S ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40S and various native Nsp1-40S and -80S complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.