RESUMEN
The emerging pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused social and economic disruption worldwide, infecting over 9.0 million people and killing over 469â000 by 24 June 2020. Unfortunately, no vaccine or antiviral drug that completely eliminates the transmissible disease coronavirus disease 2019 (COVID-19) has been developed to date. Given that coronavirus nonstructural protein 1 (nsp1) is a good target for attenuated vaccines, it is of great significance to explore the detailed characteristics of SARS-CoV-2 nsp1. Here, we first confirmed that SARS-CoV-2 nsp1 had a conserved function similar to that of SARS-CoV nsp1 in inhibiting host-protein synthesis and showed greater inhibition efficiency, as revealed by ribopuromycylation and Renilla luciferase (Rluc) reporter assays. Specifically, bioinformatics and biochemical experiments showed that by interacting with 40S ribosomal subunit, the lysine located at amino acid 164 (K164) was the key residue that enabled SARS-CoV-2 nsp1 to suppress host gene expression. Furthermore, as an inhibitor of host-protein expression, SARS-CoV-2 nsp1 contributed to cell-cycle arrest in G0/G1 phase, which might provide a favourable environment for virus production. Taken together, this research uncovered the detailed mechanism by which SARS-CoV-2 nsp1 K164 inhibited host gene expression, laying the foundation for the development of attenuated vaccines based on nsp1 modification.
Asunto(s)
Interacciones Huésped-Patógeno/genética , Lisina/genética , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , SARS-CoV-2/genética , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Biología Computacional/métodos , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Lisina/metabolismo , Mutación , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , SARS-CoV-2/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteínas no Estructurales Virales/metabolismoRESUMEN
The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3' region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2.
Asunto(s)
COVID-19/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN Viral/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Proteínas no Estructurales Virales/metabolismo , Animales , COVID-19/genética , COVID-19/patología , Chlorocebus aethiops , Microscopía por Crioelectrón , Humanos , ARN Mensajero/genética , ARN Viral/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/ultraestructura , Subunidades Ribosómicas Pequeñas de Eucariotas/virología , SARS-CoV-2/genética , SARS-CoV-2/ultraestructura , Células Vero , Proteínas no Estructurales Virales/genéticaRESUMEN
Unlike the mRNAs of their eukaryotic hosts, many RNAs of viruses lack a 5' m7GpppN cap and the 3' polyadenosine tail, and yet they are translated efficiently. Plant RNA viruses, in particular, have complex structures within their mRNA UTRs that allow them to bypass some cellular translation control steps. In the 3' UTR of maize necrotic streak virus (MNeSV), an I-shaped RNA structure (ISS) has been shown to bind eukaryotic initiation factor (eIF)4F and to mediate viral translation initiation. A 5'-3' RNA "kissing-loop" interaction is required for optimal translation. However, the details of how the 3' ISS mediates translation initiation are not well understood. Here, we studied the binding of the 3' ISS with eIFs. The eIF4A-eIF4B complex was found to increase binding affinity of eIF4F with the 3' ISS by 4-fold (from KD = 173 ± 34 nm to KD = 48 ± 11 nm). Pre-steady-state analysis indicated that the eIF4A-eIF4B complex increased the RNA association rate and decreased the dissociation rate in an ATP-independent manner. Furthermore, our findings suggest that eIF4F could promote binding of the 3' ISS with the MNeSV 5'UTR, enhancing the long-distance kissing-loop interaction. However, the association of the 5'UTR with the 3' ISS-eIF4F complex did not increase 40S ribosomal subunit binding affinity. These quantitative results suggest a stepwise model in which the first committed step is eIF4F binding to the 3' ISS, followed by an interaction with the 5'UTR and subsequent 40S ribosomal subunit binding.
Asunto(s)
Factor 4F Eucariótico de Iniciación/metabolismo , Enfermedades de las Plantas/virología , Proteínas de Plantas/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Tombusvirus/fisiología , Triticum/virología , Regiones no Traducidas 3' , Entropía , Factores Eucarióticos de Iniciación/metabolismo , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Viral/química , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/virología , Termodinámica , Triticum/metabolismoRESUMEN
The 3' untranslated region (3'UTR) of hepatitis C virus (HCV) messenger RNA stimulates viral translation by an undetermined mechanism. We identified a high affinity interaction, conserved among different HCV genotypes, between the HCV 3'UTR and the host ribosome. The 3'UTR interacts with 40S ribosomal subunit proteins residing primarily in a localized region on the 40S solvent-accessible surface near the messenger RNA entry and exit sites. This region partially overlaps with the site where the HCV internal ribosome entry site was found to bind, with the internal ribosome entry site-40S subunit interaction being dominant. Despite its ability to bind to 40S subunits independently, the HCV 3'UTR only stimulates translation in cis, without affecting the first round translation rate. These observations support a model in which the HCV 3'UTR retains ribosome complexes during translation termination to facilitate efficient initiation of subsequent rounds of translation.
Asunto(s)
Regiones no Traducidas 3' , Regulación Viral de la Expresión Génica , Hepacivirus/genética , Biosíntesis de Proteínas , Regiones no Traducidas 5' , Línea Celular Tumoral , Factor 3 de Iniciación Eucariótica/metabolismo , Humanos , ARN Viral/química , ARN Viral/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/virologíaRESUMEN
Hepatitis C virus (HCV) genome multiplication requires the concerted action of the viral RNA, host factors and viral proteins. Recent studies have provided information about the requirement of specific viral RNA motifs that play an active role in the viral life cycle. RNA regulatory motifs controlling translation and replication of the viral RNA are mostly found at the 5' and 3' untranslated regions (UTRs). In particular, viral protein synthesis is under the control of the internal ribosome entry site (IRES) element, a complex RNA structure located at the 5'UTR that recruits the ribosomal subunits to the initiator codon. Accordingly, interfering with this RNA structural motif causes the abrogation of the viral cycle. In addition, RNA translation initiation is modulated by cellular factors, including miRNAs and RNA-binding proteins. Interestingly, a RNA structural motif located at the 3'end controls viral replication and establishes long-range RNA-RNA interactions with the 5'UTR, generating functional bridges between both ends on the viral genome. In this article, we review recent advances on virus-host interaction and translation control modulating viral gene expression in infected cells.
Asunto(s)
Regulación Viral de la Expresión Génica , Genoma Viral , Hepacivirus/patogenicidad , Biosíntesis de Proteínas , ARN Viral/metabolismo , Regiones no Traducidas 5' , Antivirales/farmacología , Hepacivirus/genética , Hepacivirus/fisiología , Hepatitis C/tratamiento farmacológico , Hepatitis C/patología , Hepatitis C/virología , Humanos , Conformación de Ácido Nucleico , Motivos de Nucleótidos , ARN Viral/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/virología , Relación Estructura-Actividad , Replicación ViralRESUMEN
The eukaryotic initiation translation factor 2 (eIF2) represents a key point in the regulation of protein synthesis. This factor delivers the initiator Met-tRNA to the ribosome, a process that is conserved in all eukaryotic cells. Many types of stress reduce global translation by triggering the phosphorylation of the α subunit of eIF2, which reduces the formation of the preinitiation translation complexes. Early during rotavirus infection, eIF2α becomes phosphorylated, and even under these conditions viral protein synthesis is not affected, while most of the cell protein synthesis is blocked. Here, we found that the kinase responsible for the phosphorylation of eIF2α in rotavirus-infected cells is PKR, since in mouse embryonic fibroblasts deficient in the kinase domain of PKR, or in MA104 cells where the expression of PKR was knocked down by RNA interference, eIF2α was not phosphorylated upon rotavirus infection. The viral component responsible for the activation of PKR seems to be viral double-stranded RNA, which is found in the cytoplasm of infected cells, outside viroplasms. Taken together, these results suggest that rotaviruses induce the PKR branch of the interferon system and have evolved a mechanism to translate its proteins, surpassing the block imposed by eIF2α phosphorylation.