title: The rotavirus nonstructural protein NSP5 coordinates a [2Fe-2S] iron-sulfur cluster that modulates interaction to RNA.
abstract: During rotavirus infection, replication and packaging of the viral genome occur in viral factories, termed viroplasms. The viral nonstructural protein NSP5 is a major building block of viroplasms; it recruits the viral polymerase VP1, the core protein VP2, and the ATPase NSP2 inside the viroplasm to form the viral replication complex. Here we report that NSP5 is a unique viral metalloprotein that coordinates a [2Fe-2S] iron-sulfur cluster as demonstrated by the metal and labile sulfide contents, UV-visible light absorption, and electron paramagnetic resonance. Point mutations in NSP5 allowed us to identify C171 and C174, arranged in a CXC motif, as essential residues for cluster coordination. When coexpressed with NSP2, an NSP5 mutant devoid of the iron-sulfur cluster still forms viroplasm-like structures. The cluster is therefore neither involved in the interaction with NSP2 nor in the formation of viroplasm-like structures and thus presumably in viroplasm formation. Finally, we show using microscale thermophoresis that the iron-sulfur cluster modulates the affinity of NSP5 for single-stranded RNA. Because the cluster is near the binding sites of both the polymerase VP1 and the ATPase NSP2, we anticipate that this cluster is crucial for NSP5 functions, in either packaging or replication of the viral genome.
authors.affiliation: Virologie Moléculaire et Structurale, UPR-CNRS 3296, USC INRA, Centre de Recherche de Gif, Av de la terrasse, 91198 Gif sur Yvette, France. davy.martin@vms.cnrs-gif.fr
authors.forename: Davy
authors.initials: D
authors.lastname: Martin
authors.forename: Annie
authors.initials: A
authors.lastname: Charpilienne
authors.forename: Aubérie
authors.initials: A
authors.lastname: Parent
authors.forename: Alain
authors.initials: A
authors.lastname: Boussac
authors.forename: Benoit
authors.initials: B
authors.lastname: D'Autreaux
authors.forename: Joël
authors.initials: J
authors.lastname: Poupon
authors.forename: Didier
authors.initials: D
authors.lastname: Poncet
mesh_terms: D006801:Humans; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012401:Rotavirus / Q000737:chemistry* / Q000502:physiology; D012400:Rotavirus Infections / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology; D014779:Virus Replication / Q000502:physiology
chemical_list: D008667:Metalloproteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D013455:Sulfur; D007501:Iron