Production Of in vivo Biotinylated Rotavirus Particles

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