Development of an entirely plasmid-based reverse genetics system for 12-segmented double-stranded RNA viruses.

Abstract

The family <i>Reoviridae</i> is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family <i>Reoviridae</i>, the genera <i>Cardoreovirus</i>, <i>Phytoreovirus</i>, <i>Seadornavirus</i>, <i>Mycoreovirus</i>, and <i>Coltivirus</i> contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for <i>Reoviridae</i> viruses such as <i>Orthoreovirus</i>, <i>Orbivirus</i>, <i>Cypovirus</i>, and <i>Rotavirus</i>, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for <i>Reoviridae</i> viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus <i>Coltivirus,</i> family <i>Reoviridae</i>), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an <i>N</i>-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus <i>Coltivirus</i> but also the replication machinery of the family <i>Reoviridae</i>.