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Production of in vivo-biotinylated rotavirus particles


De Lorenzo, G; Eichwald, C; Schraner, E M; Nicolin, V; Bortul, R; Mano, M; Burrone, O R; Arnoldi, F (2012). Production of in vivo-biotinylated rotavirus particles. Journal of General Virology, 93(Pt7):1474-1482.

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.

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Institute of Veterinary Anatomy
05 Vetsuisse Faculty > Institute of Virology
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2012
Deposited On:29 Oct 2012 10:25
Last Modified:05 Apr 2016 16:02
Publisher:Society for General Microbiology
ISSN:0022-1317
Publisher DOI:https://doi.org/10.1099/vir.0.040089-0
PubMed ID:22442113

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