The cellular biology of turnip mosaic virus

正链RNA病毒的基因组复制与被感染细胞的细胞膜密切相关。例如，芜菁花叶病毒（TuMV）感染诱导在受感染的细胞中形成复杂的膜状细胞器样结构，维持病毒RNA合成和病毒细胞间运动。TuMV基因组由一个约10 000个正极性核苷酸的单个RNA分子组成。这种RNA编码一种单一的多聚蛋白，该多聚蛋白由三种病毒蛋白酶加工产生十一种成熟蛋白。一种蛋白质，即6K2蛋白，负责膜募集。在感染细胞中观察到两种6K2诱导的结构：一种大的核周球状结构，导致产生几个小的能动的外周囊泡。这些囊泡含有病毒RNA、病毒复制蛋白和宿主因子，表明6K2囊泡含有病毒复制复合物。6K2囊泡也被发现与胞间连丝相关，并可以直接移动到相邻的细胞中。因此，TuMV的共质运动作为与6K2相关的膜相关病毒复制复合物而实现。这些囊泡的细胞间运动依赖于分泌途径和肌球蛋白马达。我未来5年的研究目标将是进一步阐明这些膜重排的结构，并确定导致这些变化的一些分子事件。更准确地说，我的研究团队将使用透射电子显微镜（TEM）和断层扫描技术来获得在感染细胞和维管组织中观察到的一些细胞重塑的三维视图，特别是在木质部血管中。我们也将研究液泡膜在病毒粒子组装中的作用。我的团队还将绘制参与囊泡形成的6K2分子决定簇。初步的实验表明，6K2蛋白具有一个基于Escherichan的内质网输出信号，该信号与COPII外被体组分相互作用。我们将确认这些宿主蛋白以及SNARE样囊泡相关蛋白（VAP）AtPva11，AtPva12和AtPva22在6K2囊泡生物发生中的参与。最后，我们将使用蛋白质组学方法筛选作为6K2相关病毒复制复合物的一部分的宿主因子，并验证它们参与TuMV感染。

Genome replication of positive-strand RNA viruses is closely associated with membranes of the infected cell. For instance, turnip mosaic virus (TuMV) infection induces in the infected cells the formation of elaborate membranous organelle-like structures that sustain viral RNA synthesis and virus intercellular movement. The TuMV genome consists of a single RNA molecule of ~10 000 nucleotides of positive polarity. This RNA codes for a single polyprotein that is processed by three viral proteinases to yield eleven mature proteins. One protein, namely the 6K2 protein, is responsible for membrane recruitment. Two 6K2-induced structures are observed in infected cells: a large perinuclear globular structure that leads to the production of several small motile peripheral vesicles. These vesicles contain viral RNA, viral replication proteins and host factors, indicating that 6K2 vesicles harbour viral replication complexes. 6K2 vesicles are also found in association with plasmodesmata and can move directly into adjoining cells. Symplasmic movement of TuMV is thus achieved as a membrane-associated virus replication complex associated with 6K2. The intercellular movement of these vesicles depends on the secretory pathway and on myosin motors. My research objectives for the next 5 years will be to further elucidate the architecture of these membrane rearrangements and to identify some of the molecular events that lead to these changes. More precisely, my research team will use transmission electron microscopy (TEM) and tomography to get a 3-dimensional view of some of the cellular remodelling that are observed in infected cells and in vascular tissues, particularly in xylem vessels. We will also investigate the role of the tonoplast in virion assembly. My group will also map the molecular determinants of 6K2 that are involved in the formation of the vesicles. Preliminary experiments have indicated that the 6K2 protein has a tryptophan-based endoplasmic reticulum export signal that interacts with COPII coatomer components. We will confirm the involvement of these host proteins as well as the SNARE-like vesicle-associated protein (VAP) AtPva11, AtPva12 , and AtPva22 in 6K2 vesicle biogenesis. Finally, we will screen for host factors that are part of 6K2-associated viral replication complexes using a proteomic approach and validate their involvement in TuMV infection.