title: dsRNA binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35.
abstract: The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)-dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC(50) value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K(d) values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35-dsRNA binding functions.
authors.affiliation: Department of Life and Environmental Sciences, University of Cagliari, Cittadella di Monserrato SS554, 09042 Monserrato, Cagliari, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Zinzula
authors.forename: Francesca
authors.initials: F
authors.lastname: Esposito
authors.forename: Daniela
authors.initials: D
authors.lastname: Pala
authors.forename: Enzo
authors.initials: E
authors.lastname: Tramontano
mesh_terms: D029043:Ebolavirus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019142:Hemorrhagic Fever, Ebola / Q000821:virology*; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D012330:RNA, Double-Stranded; C080980:VP35 protein, filovirus; D054334:Viral Regulatory and Accessory Proteins