Infection of host cells by the respiratory syncytial virus (RSV) is characterized by the formation of spherical cytoplasmic inclusion bodies (IBs). These structures, which concentrate all the proteins of the polymerase complex as well as some cellular proteins, were initially considered aggresomes formed by viral dead-end products. However, recent studies revealed that IBs are viral factories where viral RNA synthesis, i.e., replication and transcription, occurs. The analysis of IBs by electron microscopy revealed that they are membrane-less structures, and accumulated data on their structure, organization, and kinetics of formation revealed that IBs share the characteristics of cellular organelles, such as P-bodies or stress granules, suggesting that their morphogenesis depends on a liquid-liquid phase separation mechanism. It was previously shown that expression of the RSV nucleoprotein N and phosphoprotein P of the polymerase complex is sufficient to induce the formation of pseudo-IBs. Here, using a series of truncated P proteins, we identified the domains of P required for IB formation and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis. We also show that pseudo-IBs can form in vitro when recombinant N and P proteins are mixed. Finally, using fluorescence recovery after photobleaching approaches, we reveal that in cellula and in vitro IBs are liquid organelles. Our results strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics.

中文翻译：

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体内和体外形成呼吸道合胞病毒胞质包涵体所需的最少元素。

呼吸道合胞病毒（RSV）感染宿主细胞的特征是形成球形胞质包涵体（IBs）。这些结构集中了聚合酶复合物的所有蛋白质以及一些细胞蛋白质，最初被认为是由病毒死角产物形成的聚集体。但是，最近的研究表明，IB是病毒RNA合成（即复制和转录）发生的病毒工厂。通过电子显微镜对IBs的分析表明，它们是无膜结构，并且积累了有关其结构，组织和形成动力学的数据，表明IBs具有细胞器的特性，例如P体或应激颗粒，这表明它们的形态取决于液-液相分离机理。先前已经表明，聚合酶复合物的RSV核蛋白N和磷蛋白P的表达足以诱导假IB的形成。在这里，我们使用一系列截短的P蛋白，鉴定了IB形成所需的P结构域，并显示了N的寡聚状态（只要它可以与RNA相互作用）对于其形态发生至关重要。我们还表明伪IB可以形成重组N和P蛋白混合后进行体外培养。最后，使用光漂白方法后的荧光恢复，我们发现在细胞中和体外IBs是液体细胞器。我们的结果有力地支持了IB的液-液相分离特性，并为进一步表征其动力学铺平了道路。