RESUMEN
Nidovirus endoribonucleases (NendoUs) include nonstructural protein 15 (nsp15) from coronaviruses and nsp11 from arteriviruses, both of which have been reported to participate in the viral replication process and in the evasion of the host immune system. Results from a previous study of coronaviruses SARS-CoV, HCoV-229E, and MHV nsp15 indicate that it mainly forms a functional hexamer, whereas nsp11 from the arterivirus PRRSV is a dimer. Here, we found that porcine Deltacoronavirus (PDCoV) nsp15 primarily exists as dimers and monomers in vitro Biological experiments reveal that a PDCoV nsp15 mutant lacking the first 27 amino acids of the N-terminal domain (Asn-1-Asn-27) forms more monomers and displays decreased enzymatic activity, indicating that this region is important for its dimerization. Moreover, multiple sequence alignments and three-dimensional structural analysis indicated that the C-terminal region (His-251-Val-261) of PDCoV nsp15 is 10 amino acids shorter and forms a shorter loop than that formed by the equivalent sequence (Gln-259-Phe-279) of SARS-CoV nsp15. This result may explain why PDCoV nsp15 failed to form hexamers. We speculate that NendoUs may have originated from XendoU endoribonucleases (XendoUs) forming monomers in eukaryotic cells, that NendoU from arterivirus gained the ability to form dimers, and that the coronavirus variants then evolved the capacity to assemble into hexamers. We further propose that PDCoV nsp15 may be an intermediate in this evolutionary process. Our findings provide a theoretical basis for improving our understanding of NendoU evolution and offer useful clues for designing drugs and vaccines against nidoviruses.
Asunto(s)
Coronavirus/química , Endorribonucleasas/química , Nidovirales/química , Subunidades de Proteína/química , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Arterivirus/química , Arterivirus/clasificación , Arterivirus/genética , Arterivirus/metabolismo , Sitios de Unión , Clonación Molecular , Coronavirus/clasificación , Coronavirus/genética , Coronavirus/metabolismo , Cristalografía por Rayos X , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Evolución Molecular , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Nidovirales/clasificación , Nidovirales/genética , Nidovirales/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/clasificación , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/genéticaRESUMEN
The nucleation of microtubules from αß-tubulin subunits is mediated by γ-tubulin complexes, which vary in composition across organisms. Aiming to understand how de novo microtubule formation is achieved and regulated by a minimal microtubule nucleation system, we here determined the cryo-electron microscopy structure of the heterotetrameric γ-tubulin small complex (γ-TuSC) from C. albicans at near-atomic resolution. Compared to the vertebrate γ-tubulin ring complex (γ-TuRC), we observed a vastly remodeled interface between the SPC/GCP-γ-tubulin spokes, which stabilizes the complex and defines the γ-tubulin arrangement. The relative positioning of γ-tubulin subunits indicates that a conformational rearrangement of the complex is required for microtubule nucleation activity, which follows opposing directionality as predicted for the vertebrate γ-TuRC. Collectively, our data suggest that the assembly and regulation mechanisms of γ-tubulin complexes fundamentally differ between the microtubule nucleation systems in lower and higher eukaryotes.
Asunto(s)
Candida albicans/metabolismo , Microtúbulos/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Tubulina (Proteína)/química , Candida albicans/química , Microscopía por Crioelectrón , Evolución Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Guanosina Difosfato/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Complejos Multiproteicos/genética , Mutación , Conformación ProteicaRESUMEN
The surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. Different coronaviruses interact with their specific receptors to enter host cells. Lentiviruses pseudotyped with their spike proteins (S) were compared to analyze the entry efficiency of various coronaviruses. Our results indicated that S proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. Furthermore, the cell tropisms of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been characterized by live and pseudotyped viruses. Both live and pseudoviruses could infected Vero- CCL-81 (monkey kidney), Huh-7 (human liver), and PK-15 (pig kidney) cells efficiently. CCL94 (cat kidney) cells could be infected efficiently by TGEV but not PEDV. Overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening.