title: Replicating reoviruses with a transgene replacing the codons for the head domain of the viral spike.
abstract: The capacity to modify the reovirus genome facilitates generation of new therapeutic reoviruses. We describe a method for generating replication-competent reoviruses carrying a heterologous transgene. The strategy is based on the expanded-tropism reovirus mutant jin-3, which can infect cells independent of the reovirus receptor junction-adhesion molecule A (JAM-A). Jin-3 harbors a mutation in the S1 segment, resulting in a G196R substitution in the tail of the spike protein σ1. The use of the jin-3 tail-encoding S1 segment allows replacing the codons for the JAM-A-binding head domain by up to 522  nucleotides of foreign sequences, without exceeding the size of the wild-type S1 segment. We inserted the codons for the porcine teschovirus-1 2A element fused with those encoding the fluorescent protein iLOV. Replicating rS1His-2A-iLOV reoviruses were generated by co-transfection of expression plasmids for all reovirus segments. These reoviruses contain the S1His-2A-iLOV segment in the absence of the wild-type S1 segment. Density-gradient centrifugation confirmed the association of the σ1-tail fragment with the capsid. Both JAM-A-positive and -negative cells exposed to the rS1His-2A-iLOV reoviruses exhibited iLOV fluorescence, confirming the jin-3-derived expanded-tropism phenotype. These data demonstrated the feasibility of generating decapitated replication-competent T3D reoviruses carrying a heterologous transgene.
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: D J M
authors.initials: DJ
authors.lastname: van den Wollenberg
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: I J C
authors.initials: IJ
authors.lastname: Dautzenberg
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: W
authors.initials: W
authors.lastname: Ros
authors.affiliation: Department of Virus Molecular Biology, Intercollegiate Faculty of Biotechnology UG-MUG, University of Gdańsk, Gdańsk, Poland.
authors.forename: A D
authors.initials: AD
authors.lastname: Lipińska
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: S K
authors.initials: SK
authors.lastname: van den Hengel
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: R C
authors.initials: RC
authors.lastname: Hoeben
mesh_terms: D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003062:Codon / Q000235:genetics*; D006224:Cricetinae; D006801:Humans; D008969:Molecular Sequence Data; D050130:Oncolytic Virotherapy; D012087:Reoviridae / Q000502:physiology*; D019076:Transgenes; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins; D003062:Codon; C031728:sigma 1 protein, reovirus