Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Laboratory of Plant Pathology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.
J Virol. 2015 Jan;89(1):480-91. doi: 10.1128/JVI.02527-14. Epub 2014 Oct 15.
Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming.
Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.
尽管许多研究已经证明了病毒蛋白或病毒复制复合物的细胞内运动，但对于它们的运动机制知之甚少。在这项研究中，我们分析了 Fig mosaic 病毒（FMV）核衣壳蛋白（NP）的定位和运动，FMV 是一种属于新近成立的 Emaravirus 属的负链 RNA 病毒。使用免疫金标记对 FMV 感染细胞的电子显微镜检查显示，NP 形成了主要被内质网（ER）膜包裹的细胞质聚集体，而未包裹的 NP 聚集体也沿着 ER 定位。同样，瞬时表达的 NPs 形成聚集体，称为 NP 体（NBs），与 ER 紧密接近，就像在 FMV 感染的细胞中一样。NP 表达细胞的亚细胞分级分离和电子显微镜分析表明，NBs 定位于细胞质中。此外，我们发现 NBs 以肌动球蛋白依赖性方式随 ER 的流动而快速移动。高浓度的布雷菲德菌素 A 处理扰乱 ER 网络结构会导致 NBs 在核周区域异常积累，表明 ER 网络结构与 NB 定位有关。XI-1、XI-2 和 XI-K 等类 XI 肌球蛋白的显性负抑制作用以相似的方式影响 ER 流动和 NB 运动。总之，这些结果表明，NBs 定位于细胞质中，但与 ER 膜紧密接近，形成包裹的颗粒，这导致细胞质 NBs 通过 ER 流动产生被动运动。
细胞内运输是病毒细胞间运动的主要和基本步骤。迄今为止，许多研究已经证明了病毒因子的快速细胞内运动，但未能提供这种运动机制或生物学意义的证据。在这里，我们观察到核衣壳蛋白（NP）的聚集体在整个细胞中快速移动，并进行了活细胞成像和超微结构分析以确定运动机制。我们提供的证据表明，植物细胞中细胞质蛋白聚集体通过肌动球蛋白介导的内质网（ER）流动被被动拖曳。在病毒感染的细胞中，NP 聚集体被 ER 膜包围，表明 NP 聚集体在靠近 ER 的位置形成包膜病毒颗粒的基础。我们的工作为植物细胞中大分子运输提供了一个复杂的模型，并提高了我们对负链 RNA 病毒包膜颗粒形成的理解。
