RESUMEN
Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a 'cap-snatching' mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/ß architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD (D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.
Asunto(s)
Endonucleasas/química , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Virus de la Coriomeningitis Linfocítica/genética , ARN Mensajero/genética , ARN Viral/genética , Transcripción Genética , Bunyaviridae/genética , Bunyaviridae/metabolismo , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Endonucleasas/genética , Endonucleasas/metabolismo , Endorribonucleasas/genética , Virus de la Coriomeningitis Linfocítica/metabolismo , Modelos Moleculares , Mutagénesis , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero/metabolismoRESUMEN
Macro domains (also called "X domains") constitute a protein module family present in all kingdoms of life, including viruses of the Coronaviridae and Togaviridae families. Crystal structures of the macro domain from the Chikungunya virus (an "Old World" alphavirus) and the Venezuelan equine encephalitis virus (a "New World" alphavirus) were determined at resolutions of 1.65 and 2.30 A, respectively. These domains are active as adenosine di-phosphoribose 1''-phosphate phosphatases. Both the Chikungunya and the Venezuelan equine encephalitis virus macro domains are ADP-ribose binding modules, as revealed by structural and functional analysis. A single aspartic acid conserved through all macro domains is responsible for the specific binding of the adenine base. Sequence-unspecific binding to long, negatively charged polymers such as poly(ADP-ribose), DNA, and RNA is observed and attributed to positively charged patches outside of the active site pocket, as judged by mutagenesis and binding studies. The crystal structure of the Chikungunya virus macro domain with an RNA trimer shows a binding mode utilizing the same adenine-binding pocket as ADP-ribose, but avoiding the ADP-ribose 1''-phosphate phosphatase active site. This leaves the AMP binding site as the sole common feature in all macro domains.