While inserting exogenous viral genome segments into rotavirus particles still remains 29 a hard challenge, here we describe the in vivo incorporation of a recombinant viral 30 capsid protein (VP6) into newly assembled rotavirus particles. We exploited the in 31 vivo biotinylation technology to biotinylate a recombinant VP6 protein fused to a 15 32 amino acid-long Biotin Acceptor Peptide (BAP), by the bacterial biotin-ligase BirA 33 contextually co-expressed in mammalian cells. To avoid toxicity of VP6 34 overexpression, we constructed a stable HEK293 cell line with tetracycline-inducible 35 expression of VP6-BAP and constitutive expression of BirA. Upon tetracycline 36 induction and rotavirus infection, VP6-BAP was biotinylated, recruited into 37 viroplasms and incorporated into newly assembled virions. The biotin molecules in 38 the capsid allowed us to use streptavidin-coated magnetic beads as a purification 39 technique alternative to CsCl gradient ultracentrifugation. Following tr

Production Of in vivo Biotinylated Rotavirus Particles

Nicolin, V.;Roberta, Bortul;Arnoldi, F.
2012-01-01

Abstract

While inserting exogenous viral genome segments into rotavirus particles still remains 29 a hard challenge, here we describe the in vivo incorporation of a recombinant viral 30 capsid protein (VP6) into newly assembled rotavirus particles. We exploited the in 31 vivo biotinylation technology to biotinylate a recombinant VP6 protein fused to a 15 32 amino acid-long Biotin Acceptor Peptide (BAP), by the bacterial biotin-ligase BirA 33 contextually co-expressed in mammalian cells. To avoid toxicity of VP6 34 overexpression, we constructed a stable HEK293 cell line with tetracycline-inducible 35 expression of VP6-BAP and constitutive expression of BirA. Upon tetracycline 36 induction and rotavirus infection, VP6-BAP was biotinylated, recruited into 37 viroplasms and incorporated into newly assembled virions. The biotin molecules in 38 the capsid allowed us to use streptavidin-coated magnetic beads as a purification 39 technique alternative to CsCl gradient ultracentrifugation. Following tr
Pubblicato
http://www.ncbi.nlm.nih.gov/pubmed?term=arnoldi de lorenzo
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/2503543
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? 3
  • Scopus 5
  • ???jsp.display-item.citation.isi??? 5
social impact