Production of in vivo-biotinylated rotavirus particles

De Lorenzo, G. , Eichwald, C., Schraner, E.M., Nicolin, V., Bortul, R., Mano, M., Burrone, O.R. and Arnoldi, F. (2012) Production of in vivo-biotinylated rotavirus particles. Journal of General Virology, 93(7), pp. 1474-1482. (doi: 10.1099/vir.0.040089-0) (PMID:22442113)

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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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:De Lorenzo, Dr Giuditta
Authors: De Lorenzo, G., Eichwald, C., Schraner, E.M., Nicolin, V., Bortul, R., Mano, M., Burrone, O.R., and Arnoldi, F.
College/School:College of Medical Veterinary and Life Sciences > School of Infection & Immunity
College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
Journal Name:Journal of General Virology
Publisher:Microbiology Society
ISSN:0022-1317
ISSN (Online):1465-2099

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