title: A plasmid-based reverse genetics system for mammalian orthoreoviruses driven by a plasmid-encoded T7 RNA polymerase.
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.
authors.affiliation: Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan. Electronic address: satoshik@fujita-hu.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Komoto
authors.affiliation: Laboratory of Viral Replication, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases (BIKEN), Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kawagishi
authors.affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, United States; Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, TN 37232, United States; Laboratory of Viral Replication, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases (BIKEN), Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, United States; Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, TN 37232, United States.
authors.forename: Mine
authors.initials: M
authors.lastname: Ikizler
authors.affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, United States; Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, TN 37232, United States.
authors.forename: Jason
authors.initials: J
authors.lastname: Iskarpatyoti
authors.affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, United States; Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, United States; Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, TN 37232, United States.
authors.forename: Terence S
authors.initials: TS
authors.lastname: Dermody
authors.affiliation: Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Taniguchi
mesh_terms: D000818:Animals; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006801:Humans; D030102:Orthoreovirus, Mammalian / Q000235:genetics*; D059386:Reverse Genetics / Q000379:methods*; D014162:Transfection; D014764:Viral Proteins / Q000378:metabolism*; D014773:Virology / Q000379:methods*
chemical_list: D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: Eukaryotic expression plasmid; Reovirus; Reverse genetics; T7 RNA polymerase