Origins of Enterovirus Replication Organelles Established by Whole-Cell Electron Microscopy
Origins of Enterovirus Replication Organelles Established by Whole-Cell Electron Microscopy
复制标题
DOI：
10.1128/mbio.00951-19
复制
发表时间：
2019-05-01
期刊：
影响因子：
6.4
通讯作者：
Barcena, Montserrat
中科院分区：
文献类型：
--
作者：
Melia, Charlotte E.;Peddie, Christopher J.;Barcena, Montserrat
肠道病毒基因组复制发生在病毒诱导的细胞膜和脂质结构上。然而，这些复制细胞器（RO）的起源仍然不确定。缺乏膜供体的超微结构证据，表明其转变为RO的位点是罕见的或短暂的。为了克服这一挑战，我们结合了活细胞成像和全细胞的连续块面扫描电子显微镜来捕获新出现的肠道病毒RO。荧光标记的病毒蛋白的第一个病灶与连接到内质网（ER）的RO相关，并且在来自trans-Golgi网络的RO出现之前。全细胞数据集进一步揭示了RO和脂滴之间的显著接触区域，这可能代表了脂质穿梭以促进RO增殖和基因组复制的途径。我们的数据提供了直接的证据，肠道病毒使用ER，然后高尔基膜启动RO形成，展示了显着的灵活性，肠道病毒篡夺细胞器。重要性肠道病毒是一系列人类疾病的病原体。这些病毒在细胞内的复制依赖于被称为复制细胞器（RO）的专门的膜结构，其在感染期间形成，但其起源仍然难以捉摸。为了捕捉肠道病毒RO的出现，我们使用相关的光和连续块面扫描电子显微镜，一个强大的方法来查明罕见的事件，在他们的全细胞超微结构的背景。RO生物发生首先发生在ER，然后在高尔基体膜。早期RO和脂滴（LD）之间存在广泛的接触，这可能有助于提供复制所需的LD衍生脂质。总之，这些数据建立了肠道病毒RO的双重起源和它们在不同支持细胞膜上的生物发生年表。
Enterovirus genome replication occurs at virus-induced structures derived from cellular membranes and lipids. However, the origin of these replication organelles (ROs) remains uncertain. Ultrastructural evidence of the membrane donor is lacking, suggesting that the sites of its transition into ROs are rare or fleeting. To overcome this challenge, we combined live-cell imaging and serial block-face scanning electron microscopy of whole cells to capture emerging enterovirus ROs. The first foci of fluorescently labeled viral protein correlated with ROs connected to the endoplasmic reticulum (ER) and preceded the appearance of ROs stemming from the trans-Golgi network. Whole-cell data sets further revealed striking contact regions between ROs and lipid droplets that may represent a route for lipid shuttling to facilitate RO proliferation and genome replication. Our data provide direct evidence that enteroviruses use ER and then Golgi membranes to initiate RO formation, demonstrating the remarkable flexibility with which enteroviruses usurp cellular organelles.IMPORTANCE Enteroviruses are causative agents of a range of human diseases. The replication of these viruses within cells relies on specialized membranous structures termed replication organelles (ROs) that form during infection but whose origin remains elusive. To capture the emergence of enterovirus ROs, we use correlative light and serial block-face scanning electron microscopy, a powerful method to pinpoint rare events in their whole-cell ultrastructural context. RO biogenesis was found to occur first at ER and then at Golgi membranes. Extensive contacts were found between early ROs and lipid droplets (LDs), which likely serve to provide LD-derived lipids required for replication. Together, these data establish the dual origin of enterovirus ROs and the chronology of their biogenesis at different supporting cellular membranes.