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1.
Nucleic Acids Res ; 48(10): 5749-5765, 2020 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-32313945

RESUMEN

The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, is an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron cryo-microscopy and X-ray crystallography, we obtained 3D models covering ∼70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein-RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , Phlebovirus/enzimología , Proteínas Virales/química , Microscopía por Crioelectrón , ARN Polimerasas Dirigidas por ADN/metabolismo , Endorribonucleasas/metabolismo , Modelos Moleculares , Phlebovirus/genética , Regiones Promotoras Genéticas , Dominios Proteicos , Virus ARN/enzimología , Proteínas Virales/metabolismo , Replicación Viral
2.
Cell Rep ; 30(1): 153-163.e5, 2020 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-31914382

RESUMEN

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus with 12%-30% case mortality rates and is related to the Heartland virus (HRTV) identified in the United States. Together, SFTSV and HRTV are emerging segmented, negative-sense RNA viral (sNSV) pathogens with potential global health impact. Here, we characterize the amino-terminal cap-snatching endonuclease domain of SFTSV polymerase (L) and solve a 2.4-Å X-ray crystal structure. While the overall structure is similar to those of other cap-snatching sNSV endonucleases, differences near the C terminus of the SFTSV endonuclease suggest divergence in regulation. Influenza virus endonuclease inhibitors, including the US Food and Drug Administration (FDA) approved Baloxavir (BXA), inhibit the endonuclease activity in in vitro enzymatic assays and in cell-based studies. BXA displays potent activity with a half maximal inhibitory concentration (IC50) of ∼100 nM in enzyme inhibition and an EC50 value of ∼250 nM against SFTSV and HRTV in plaque assays. Together, our data support sNSV endonucleases as an antiviral target.


Asunto(s)
Antivirales/farmacología , Endonucleasas/química , Phlebovirus/efectos de los fármacos , Phlebovirus/enzimología , Animales , Antivirales/química , Cationes Bivalentes/farmacología , Línea Celular , Secuencia Conservada , Cristalografía por Rayos X , Dibenzotiepinas/química , Dibenzotiepinas/farmacología , Endonucleasas/antagonistas & inhibidores , Endonucleasas/metabolismo , Humanos , Modelos Moleculares , Morfolinas/química , Morfolinas/farmacología , Dominios Proteicos , Estructura Secundaria de Proteína , Piridonas/química , Piridonas/farmacología , Triazinas/química , Triazinas/farmacología
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