Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed “duality” characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.

与双链 DNA (dsDNA)、单链 DNA (ssDNA) 和 ssRNA 病毒不同，dsRNA 病毒的基因组包装机制知之甚少。在这里，我们结合了高分辨率冷冻电子显微镜（cryo-EM）、细胞冷冻电子断层扫描（cryo-ET）和结构引导诱变技术来研究蓝舌病毒（BTV）的基因组包装和衣壳组装，蓝舌病毒是蓝舌病毒的成员。dsRNA 病毒呼肠孤病毒科。总共捕获了 BTV 衣壳的 11 种组装状态，分辨率高达 2.8 Å，大部分在宿主细胞质中可见。 ATPase VP6 被发现位于衣壳壳蛋白 VP3 的顶点下方，作为一种含有 RNA 的五聚体，促进 RNA 包装。 RNA 包装扩展了 VP3 外壳，然后与中层和外层蛋白质结合以产生感染性病毒粒子。这些揭示的 BTV 组装机制的“二元性”特征调和了先前相互矛盾的共组装和核心填充模型，并为呼肠孤病毒科成员及其他成员的神秘 RNA 包装和衣壳组装提供了见解。