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  • Original Article - Enabling Technologies
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Original Article – Enabling Technologies

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

Gene Therapy volume 22, pages 267–279 (2015)Cite this article

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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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Acknowledgements

We thank Dr Danijela Koppers-Lalic and Leithe Buedel for helpful discussions and suggestions. Aitor Navarro is gratefully acknowledged for providing us the protocol for RNA electrophoresis and helpful comments.

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Authors and Affiliations

  1. Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands

    D J M van den Wollenberg, I J C Dautzenberg, W Ros, S K van den Hengel & R C Hoeben

  2. Department of Virus Molecular Biology, Intercollegiate Faculty of Biotechnology UG-MUG, University of Gdańsk, Gdańsk, Poland

    A D Lipińska

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  1. D J M van den Wollenberg
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Correspondence to R C Hoeben.

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van den Wollenberg, D., Dautzenberg, I., Ros, W. et al. Replicating reoviruses with a transgene replacing the codons for the head domain of the viral spike. Gene Ther 22, 267–279 (2015). https://doi.org/10.1038/gt.2014.126

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