A plasmid-based reverse genetics system for mammalian orthoreoviruses driven by a plasmid-encoded T7 RNA polymerase

Satoshi Komoto, Takahiro Kawagishi, Takeshi Kobayashi, Mine Ikizler, Jason Iskarpatyoti, Terence S. Dermody, Koki Taniguchi

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Mammalian orthoreoviruses (reoviruses) have served as highly useful models for studies of virus replication and pathogenesis. The development of a plasmid-based reverse genetics system represented a major breakthrough in reovirus research. The current reverse genetics systems for reoviruses rely on the expression of T7 RNA polymerase within cells transfected with reovirus gene-segment cDNA plasmids. In these systems, the T7 RNA polymerase is provided by using a recombinant vaccinia virus expressing T7 RNA polymerase or a cell line constitutively expressing T7 RNA polymerase. Here, we describe an alternative plasmid-based rescue system driven by a plasmid-encoded T7 RNA polymerase, which could increase the flexibility of such reverse genetics systems. Although this approach requires transfection of an additional plasmid, virus recovery was achieved when A549, BHK-21, or L929 cells were co-transfected with a reovirus 10-plasmid set together with a plasmid encoding T7 RNA polymerase. Theoretically, this system offers the possibility to generate reoviruses in any cell line, including those amenable to propagation of viral vectors for clinical use. Thus, this approach will increase the flexibility of reverse genetics for basic studies of reovirus biology and foster development of reoviruses for clinical applications.

Original languageEnglish
Pages (from-to)36-39
Number of pages4
JournalJournal of Virological Methods
Volume196
DOIs
Publication statusPublished - 01-02-2014

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Mammalian Orthoreovirus
Reverse Genetics
Plasmids
Cell Line
Vaccinia virus
Virus Replication
bacteriophage T7 RNA polymerase
Transfection
Complementary DNA
Viruses

All Science Journal Classification (ASJC) codes

  • Virology

Cite this

Komoto, Satoshi ; Kawagishi, Takahiro ; Kobayashi, Takeshi ; Ikizler, Mine ; Iskarpatyoti, Jason ; Dermody, Terence S. ; Taniguchi, Koki. / A plasmid-based reverse genetics system for mammalian orthoreoviruses driven by a plasmid-encoded T7 RNA polymerase. In: Journal of Virological Methods. 2014 ; Vol. 196. pp. 36-39.
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A plasmid-based reverse genetics system for mammalian orthoreoviruses driven by a plasmid-encoded T7 RNA polymerase. / Komoto, Satoshi; Kawagishi, Takahiro; Kobayashi, Takeshi; Ikizler, Mine; Iskarpatyoti, Jason; Dermody, Terence S.; Taniguchi, Koki.

In: Journal of Virological Methods, Vol. 196, 01.02.2014, p. 36-39.

Research output: Contribution to journalArticle

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