Abdelhakim Aliaa H, Salgado Eric N, Fu Xiaofeng, Pasham Mithun, Nicastro Daniela, Kirchhausen Tomas, Harrison Stephen C
Laboratory of Molecular Medicine, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts, United States of America.
PLoS Pathog. 2014 Sep 11;10(9):e1004355. doi: 10.1371/journal.ppat.1004355. eCollection 2014 Sep.
Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex--for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼ 700 Å inner capsid particle ("double-layered particle", DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼ 10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.
无包膜病毒进入细胞需要将一个大分子复合物转运到细胞质中——对于双链RNA病毒来说，是一个完整的亚病毒颗粒。我们利用活细胞荧光成像技术追踪轮状病毒进入并穿透其约700埃的内衣壳颗粒（“双层颗粒”，DLP）的细胞质的过程。我们用不同的荧光标签标记DLP和两种外层蛋白中的每一种，并追踪这些颗粒在与BSC-1细胞结合并进入细胞时每种物质的命运。病毒粒子附着在宿主细胞膜上的糖脂受体上，并迅速对外源添加物变得不可接近；通过DLP远离残留外层蛋白的快速扩散运动检测到，大多数将其DLP释放到细胞质中的颗粒在约10分钟时就已完成。电子显微镜显示了颗粒被紧密包裹的膜内陷吞噬的各个阶段的图像，这与轮状病毒颗粒驱动自身摄取的解释一致。膜结合病毒粒子的电子冷冻断层扫描也显示了紧密包裹的膜。结合有关病毒成分的高分辨率结构信息，这些观察结果提出了一个膜破坏和DLP穿透的分子模型。
