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. 2015 Mar;22(3):267-79.
doi: 10.1038/gt.2014.126. Epub 2015 Jan 15.

Replicating reoviruses with a transgene replacing the codons for the head domain of the viral spike

D J M van den Wollenberg  1 , I J C Dautzenberg  1 , W Ros  1 , A D Lipińska  2 , S K van den Hengel  1 , R C Hoeben  1
Affiliations

Replicating reoviruses with a transgene replacing the codons for the head domain of the viral spike

D J M van den Wollenberg et al. Gene Ther. 2015 Mar.

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.

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