title: Production of in vivo-biotinylated rotavirus particles.
abstract: Although inserting exogenous viral genome segments into rotavirus particles remains a hard challenge, this study describes the in vivo incorporation of a recombinant viral capsid protein (VP6) into newly assembled rotavirus particles. In vivo biotinylation technology was exploited to biotinylate a recombinant VP6 protein fused to a 15 aa biotin-acceptor peptide (BAP) by the bacterial biotin ligase BirA contextually co-expressed in mammalian cells. To avoid toxicity of VP6 overexpression, a stable HEK293 cell line was constructed with tetracycline-inducible expression of VP6-BAP and constitutive expression of BirA. Following tetracycline induction and rotavirus infection, VP6-BAP was biotinylated, recruited into viroplasms and incorporated into newly assembled virions. The biotin molecules in the capsid allowed the use of streptavidin-coated magnetic beads as a purification technique instead of CsCl gradient ultracentrifugation. Following transfection, double-layered particles attached to beads were able to induce viroplasm formation and to generate infective viral progeny.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34149 Trieste, Italy.
authors.forename: G
authors.initials: G
authors.lastname: De Lorenzo
authors.affiliation: Institute of Virology, University of Zürich, Winterthurerstrasse 260, CH-8057 Zürich, Switzerland.
authors.forename: C
authors.initials: C
authors.lastname: Eichwald
authors.affiliation: Institute of Veterinary Anatomy, University of Zürich, Winterthurerstrasse 260, CH-8057 Zürich, Switzerland.|Institute of Virology, University of Zürich, Winterthurerstrasse 260, CH-8057 Zürich, Switzerland.
authors.forename: E M
authors.initials: EM
authors.lastname: Schraner
authors.affiliation: Dipartimento Universitario Clinico di Scienze Mediche, Chirurgiche e della Salute, Strada di Fiume 447, 34149 Trieste, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Nicolin
authors.affiliation: Dipartimento Universitario Clinico di Scienze Mediche, Chirurgiche e della Salute, Strada di Fiume 447, 34149 Trieste, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Bortul
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34149 Trieste, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Mano
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34149 Trieste, Italy.
authors.forename: O R
authors.initials: OR
authors.lastname: Burrone
authors.affiliation: Dipartimento Universitario Clinico di Scienze Mediche, Chirurgiche e della Salute, Strada di Fiume 447, 34149 Trieste, Italy.|International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34149 Trieste, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Arnoldi
mesh_terms: D019921:Biotinylation / Q000379:methods*; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012401:Rotavirus / Q000235:genetics / Q000254:growth & development* / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods*; D014773:Virology / Q000379:methods*; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D011994:Recombinant Proteins