title: Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery.
abstract: Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure. Using this insight, we exploited the unencumbered pore at the 5-fold axis of symmetry and the absence of encapsidated RNA to label the interior of empty CLPs with a fluorescent bioconjugate. CLPs containing 120 GFP molecules and those containing approximately 150 dye molecules were both shown to bind human integrin via a naturally occurring Arg-Gly-Asp motif found on an exposed loop of the VP7 trimeric spike. Furthermore, fluorescently labeled CLPs were shown to interact with a cell line overexpressing the surface receptor. Thus, BTV CLPs present themselves as a useful tool in targeted cargo delivery. These results highlight the importance of detailed structural analysis of VNPs in validating their molecular organization and the value of such analyses in aiding their design and further modification.
authors.forename: Lou
authors.initials: L
authors.lastname: Brillault
authors.forename: Philippe V
authors.initials: PV
authors.lastname: Jutras
authors.forename: Noor
authors.initials: N
authors.lastname: Dashti
authors.affiliation: Department of Biological Chemistry, John Innes Centre , Norwich Research Park, Colney, Norfolk NR4 7UH, United Kingdom.
authors.forename: Eva C
authors.initials: EC
authors.lastname: Thuenemann
authors.forename: Garry
authors.initials: G
authors.lastname: Morgan
authors.affiliation: Department of Biological Chemistry, John Innes Centre , Norwich Research Park, Colney, Norfolk NR4 7UH, United Kingdom.
authors.forename: George P
authors.initials: GP
authors.lastname: Lomonossoff
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Landsberg
authors.forename: Frank
authors.identifier: 0000-0001-8152-3820
authors.initials: F
authors.lastname: Sainsbury
mesh_terms: D001820:Bluetongue virus / Q000737:chemistry; D003001:Cloning, Molecular; D004337:Drug Carriers / Q000737:chemistry; D016503:Drug Delivery Systems; D006801:Humans; D016023:Integrins / Q000737:chemistry; D061986:MCF-7 Cells; D053758:Nanoparticles / Q000737:chemistry*; D036103:Nanotechnology; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification; D014026:Nicotiana / Q000737:chemistry*
chemical_list: D004337:Drug Carriers; D016023:Integrins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins
keywords: bioconjugation; bluetongue virus; core-like particle; cryo-EM; guest protein encapsidation; receptor targeting; virus-like nanoparticle