Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom.
Proc Natl Acad Sci U S A. 2011 Aug 16;108(33):13746-51. doi: 10.1073/pnas.1108667108. Epub 2011 Aug 1.
Bluetongue virus (BTV) is a vector-borne, nonenveloped icosahedral particle that is organized in two capsids, an outer capsid of two proteins, VP2 and VP5, and an inner capsid (or core) composed of two major proteins, VP7 and VP3, in two layers. The VP3 layer (subcore) encloses viral transcription complex (VP1 polymerase, VP4 capping enzyme, VP6 helicase) and a 10-segmented double-stranded (dsRNA) genome. Although much is known about the BTV capsids, the order of the core assembly and the mechanism of genome packaging remain unclear. Here, we established a cell-free system to reconstitute subcore and core structures with the proteins and ssRNAs, demonstrating that reconstituted cores are infectious in insect cells. Furthermore, we showed that the BTV ssRNAs are essential to drive the assembly reaction and that there is a distinct order of internal protein recruitment during the assembly process. The in vitro engineering of infectious BTV cores is unique for any member of the Reoviridae and will facilitate future studies of RNA-protein interactions during BTV core assembly.
蓝舌病毒（BTV）是一种经媒介传播的、无包膜的二十面体颗粒，由两个衣壳组成，外壳由两种蛋白 VP2 和 VP5 组成，内壳（或核心）由两种主要蛋白 VP7 和 VP3 组成，分为两层。VP3 层（亚核）包含病毒转录复合物（VP1 聚合酶、VP4 加帽酶、VP6 解旋酶）和由 10 个片段组成的双链 RNA（dsRNA）基因组。尽管人们对 BTV 衣壳有了很多了解，但核心组装的顺序和基因组包装的机制仍不清楚。在这里，我们建立了一个无细胞系统，用蛋白质和 ssRNA 重新组装亚核和核心结构，证明了组装的核心在昆虫细胞中具有感染性。此外，我们还表明，BTV ssRNA 对驱动组装反应至关重要，并且在组装过程中存在内部蛋白募集的明显顺序。体外工程化的感染性 BTV 核心对于呼肠孤病毒科的任何成员都是独特的，这将有助于未来研究 BTV 核心组装过程中的 RNA-蛋白相互作用。
