title: Purification and functional characterization of the full length recombinant Ebola virus VP35 protein expressed in E. coli.
abstract: In this work is presented, for the first time, the expression and purification in a prokaryotic system of the functionally active, recombinant full length VP35 protein of Ebola virus (EBOV). EBOV is an enveloped non-segmented negative-stranded RNA virus belonging to the filovirus family which causes a severe hemorrhagic fever in humans with mortality rates as high as 90%. Several lines of evidence suggest that EBOV interferes with host interferon responses and that the lack of these responses allows its rapidly progressive, overwhelming infection. Recently, the EBOV-encoded VP35 protein, essential cofactor of the viral RNA polymerase complex, has been shown to play an important role as interferon antagonist and the structure of his C-terminal IFN inhibitory domain has been solved. Although it is clearly important to better understand VP35 biochemical functions and its interplay with viral and cellular factors, the attempts to obtain full length E. coli recombinant VP35 (rVP35) have, until now, failed. In this study, we expressed the full length EBOV VP35 in E. coli as a soluble N-terminal His(6)-tag fusion protein and purified it to >95% homogeneity. In order to compare native and rVP35 functions, we characterized the rVP35 for its homo-oligomeric status and its RNA binding capacity showing that bacterially expressed rVP35 has the same properties as VP35 expressed in eukaryotic cells and that, therefore, rVP35 can be used as a valid model for functional studies and the validation of biochemical assays aimed to identify antiviral inhibitors which can interfere with the EBOV replication cycle.
authors.affiliation: Department of Applied Sciences in Biosystems, University of Cagliari, Cittadella di Monserrato SS554, Monserrato, Cagliari, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Zinzula
authors.forename: Francesca
authors.initials: F
authors.lastname: Esposito
authors.forename: Elke
authors.initials: E
authors.lastname: Mühlberger
authors.forename: Martina
authors.initials: M
authors.lastname: Trunschke
authors.forename: Dominik
authors.initials: D
authors.lastname: Conrad
authors.forename: Dario
authors.initials: D
authors.lastname: Piano
authors.forename: Enzo
authors.initials: E
authors.lastname: Tramontano
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007372:Interferons / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; C080980:VP35 protein, filovirus; D054334:Viral Regulatory and Accessory Proteins; D007372:Interferons