Development of an entirely plasmid-based reverse genetics system for 12-segmented double-stranded RNA viruses
Development of an entirely plasmid-based reverse genetics system for 12-segmented double-stranded RNA viruses
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DOI：
10.1073/pnas.2105334118
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发表时间：
2021-10-19
影响因子：
11.1
通讯作者：
Kobayashi, Takeshi
中科院分区：
文献类型：
--
作者：
Nouda, Ryotaro;Minami, Shohei;Kobayashi, Takeshi
呼肠孤病毒科是具有包含9至12个区段的双链（ds）RNA基因组的无包膜病毒组。在呼肠孤病毒科中，Cardoreovirus、Phytoreovirus、Seadornavirus、Mycoreovirus和Coltivirus属含有具有12节段dsRNA基因组的病毒物种。用于产生重组感染性病毒的反向遗传学系统是研究病毒基因功能以及开发疫苗和治疗干预的有力工具。通常，该方法已用于呼肠孤病毒科病毒，如正呼肠孤病毒、环状病毒、Cypovirus和轮状病毒，其基因组分别具有10或11个区段。然而，尚未开发出用于具有携带12个区段的基因组的呼肠孤病毒科病毒的反向遗传学系统。在这里，我们描述了一个完整的基于质粒的反向遗传学系统的Tarumizu蜱病毒（TarTV）（Coltivirus属，呼肠孤病毒科），它有一个12节段的基因组的发展。通过将编码TarTV基因组的12个克隆互补DNA转染到表达T7 RNA聚合酶的幼仓鼠肾细胞中来产生重组TarTV。利用这种技术，我们产生了VP 12突变体病毒，并证明VP 12是一种N-糖基化蛋白。我们还产生了表达HiBiT标记的VP 8蛋白的报告病毒。这种反向遗传学系统不仅将增加我们对Coltivirus属的生物学的理解，还将增加我们对呼肠孤病毒科的复制机制的理解。
The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus 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 Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae 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 Coltivirus, family Reoviridae), 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 N-glycosylated protein. We also generated a reporter virus expressing the HiBiTtagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.