Intracellular Colocalization of Influenza Viral RNA and Rab11A Is Dependent upon Microtubule Filaments.
Intracellular Colocalization of Influenza Viral RNA and Rab11A Is Dependent upon Microtubule Filaments.
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DOI：
10.1128/jvi.01179-17
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发表时间：
2017-10-01
影响因子：
5.4
通讯作者：
Lakdawala SS
中科院分区：
文献类型：
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作者：
Nturibi E;Bhagwat AR;Coburn S;Myerburg MM;Lakdawala SS
甲型流感病毒 (IAV) 由八个病毒 RNA (vRNA) 片段组成，这些片段在宿主细胞核中复制并转运到质膜以包装成子代病毒颗粒。我们之前提出了一个模型，其中 vRNA 子复合物从细胞核输出并在到达质膜的途中组装。然而，宿主细胞骨架蛋白在 IAV vRNA 片段的细胞质组装中的作用仍然未知。先前的研究表明，IAV vRNA 片段通过含有 Rab11A 的回收内体 (RE) 进行运输，并使用微管 (MT) 和肌动蛋白。 Rab11A RE 主要沿 MT 运输；因此，有必要研究 MT 在 vRNA 组装中的作用。我们通过使用多种细胞类型（包括原代人气道上皮细胞）中的多种 IAV 毒株，探索了 MT 在 vRNA 组装和复制中的作用。我们观察到，在 MT 解聚药物存在的情况下，Rab11A 的定位发生了改变，但 IAV 在所有测试的细胞类型中的生长没有变化。进行荧光原位杂交以确定 MT 在多个 vRNA 片段组装中的作用。出乎意料的是，我们发现细胞质灶中的 vRNA-vRNA 关联与 MT 无关。鉴于在缺乏完整 MT 丝的情况下 Rab11A 和 vRNA 片段之间的定位差异，我们分析了感染细胞细胞质中 Rab11A 和 vRNA 之间的三维空间关系。我们发现 Rab11A 和 vRNA 共定位依赖于动态 MT 丝。综上所述，我们的数据表明流感 vRNA 的细胞质转运可能包括 Rab11A RE 独立机制。重要性 IAV 感染通过季节性流行病和散发性流行病造成巨大的公共卫生负担。流行性 IAV 是通过动物或人类宿主体内 vRNA 的重排而出现的。阐明 IAV 组装的细胞内动力学机制对于理解重排是必要的。我们描述 MT 在 vRNA 运输和组装中的作用的结果扩展了先前表征 vRNA 组装的研究。这项研究首次评估了 MT 在人类支气管气道上皮细胞中流感病毒复制中的作用。此外，我们还提供了关于 MT 在促进不同 vRNA 片段之间关联方面的作用的新数据。有趣的是，我们的结果表明，vRNA 片段的渐进组装可能依赖于细胞类型，并且在缺乏完整 MT 的情况下，vRNA 可以在没有 Rab11A RE 的情况下通过细胞质转运。这些结果增强了我们对 vRNA 组装以及细胞骨架蛋白在该过程中的作用的理解。
Influenza A virus (IAV) consists of eight viral RNA (vRNA) segments that are replicated in the host cell nucleus and transported to the plasma membrane for packaging into progeny virions. We have previously proposed a model where subcomplexes of vRNA are exported from the nucleus and assembled en route to the plasma membrane. However, the role of host cytoskeletal proteins in the cytoplasmic assembly of IAV vRNA segments remains unknown. Previous studies have suggested that IAV vRNA segments are transported via Rab11A-containing recycling endosomes (RE) and use both microtubules (MT) and actin. Rab11A RE transport primarily along MT; therefore, investigation of the role of MT in vRNA assembly is warranted. We explored the role of MT in vRNA assembly and replication by using multiple IAV strains in various cell types, including primary human airway epithelial cells. We observed that Rab11A localization was altered in the presence of MT-depolymerizing drugs, but growth of IAV in all of the cell types tested was unchanged. Fluorescent in situ hybridization was performed to determine the role of MT in the assembly of multiple vRNA segments. Unexpectedly, we found that vRNA-vRNA association in cytoplasmic foci was independent of MT. Given the disparity of localization between Rab11A and vRNA segments in the absence of intact MT filaments, we analyzed the three-dimensional spatial relationship between Rab11A and vRNA in the cytoplasm of infected cells. We found that Rab11A and vRNA colocalization is dependent upon dynamic MT filaments. Taken together, our data suggest that cytoplasmic transport of influenza vRNA may include a Rab11A RE-independent mechanism. IMPORTANCE IAV infections cause a large public health burden through seasonal epidemics and sporadic pandemics. Pandemic IAVs emerge through reassortment of vRNA in animal or human hosts. Elucidation of the mechanism of intracellular dynamics of IAV assembly is necessary to understand reassortment. Our results describing the role of MT in vRNA transport and assembly expand upon previous studies characterizing vRNA assembly. This study is the first to assess the role of MT in influenza virus replication in human bronchial airway epithelial cells. In addition, we present novel data on the role of MT in facilitating the association between distinct vRNA segments. Interestingly, our results suggest that progressive assembly of vRNA segments may be cell type dependent and that vRNA may be transported through the cytoplasm without Rab11A RE in the absence of intact MT. These results enhance our understanding of vRNA assembly and the role of cytoskeletal proteins in that process.