Abstract：
Phase separation has emerged as a fundamental principle for organizing viral and cellular membraneless organelles. Although these subcellular compartments have been recognized for decades, their biogenesis and mechanisms of regulation are poorly understood. Here, we investigate the formation of membraneless inclusion bodies (IBs) induced during the infection of a plant rhabdovirus, tomato yellow mottle-associated virus (TYMaV). We generated recombinant TYMaV encoding a fluorescently labeled IB constituent protein and employed live-cell imaging to characterize the intracellular dynamics and maturation of viral IBs in infected Nicotiana benthamiana cells. We show that TYMaV IBs are phase-separated biomolecular condensates and that viral nucleoprotein and phosphoprotein are minimally required for IB formation in vivo and in vitro. TYMaV IBs move along the microfilaments, likely through the anchoring of viral phosphoprotein to myosin XIs. Furthermore, pharmacological disruption of microfilaments or inhibition of myosin XI functions suppresses IB motility, resulting in arrested IB growth and inefficient virus replication. Our study establishes phase separation as a process driving the formation of liquid viral factories and emphasizes the role of the cytoskeletal system in regulating the dynamics of condensate maturation.
摘要：
相分离已成为组织病毒和细胞无膜细胞器的基本原理。尽管这些亚细胞区室已经被认识了几十年，它们的生物发生和调节机制知之甚少。这里,我们研究了在植物弹状病毒感染过程中诱导的无膜包涵体（IBs）的形成，番茄黄斑驳相关病毒（TYMaV）。我们产生了编码荧光标记的IB组成蛋白的重组TYMaV，并采用活细胞成像来表征感染的本氏烟草细胞中病毒IBs的细胞内动力学和成熟。我们表明TYMaVIB是相分离的生物分子缩合物，并且病毒核蛋白和磷蛋白是体内和体外IB形成所需的最低限度。TYMaVIB沿着微丝移动，可能是通过将病毒磷蛋白锚定到肌球蛋白XIs。此外，微丝的药理破坏或肌球蛋白XI功能的抑制抑制IB运动，导致IB生长停滞和病毒复制效率低下。我们的研究将相分离确立为驱动液体病毒工厂形成的过程，并强调细胞骨架系统在调节冷凝物成熟动力学中的作用。
