Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
Department of Microbiology, University of Chicago, Chicago, Illinois, United States of America.
PLoS Pathog. 2021 Sep 2;17(9):e1009321. doi: 10.1371/journal.ppat.1009321. eCollection 2021 Sep.
Influenza A virus [IAV] genomes comprise eight negative strand RNAs packaged into virions in the form of viral ribonucleoproteins [vRNPs]. Rab11a plays a crucial role in the transport of vRNPs from the nucleus to the plasma membrane via microtubules, allowing assembly and virus production. Here, we identify a novel function for Rab11a in the inter-cellular transport of IAV vRNPs using tunneling nanotubes [TNTs]as molecular highways. TNTs are F-Actin rich tubules that link the cytoplasm of nearby cells. In IAV-infected cells, Rab11a was visualized together with vRNPs in these actin-rich intercellular connections. To better examine viral spread via TNTs, we devised an infection system in which conventional, virion-mediated, spread was not possible. Namely, we generated HA-deficient reporter viruses which are unable to produce progeny virions but whose genomes can be replicated and trafficked. In this system, vRNP transfer to neighboring cells was observed and this transfer was found to be dependent on both actin and Rab11a. Generation of infectious virus via TNT transfer was confirmed using donor cells infected with HA-deficient virus and recipient cells stably expressing HA protein. Mixing donor cells infected with genetically distinct IAVs furthermore revealed the potential for Rab11a and TNTs to serve as a conduit for genome mixing and reassortment in IAV infections. These data therefore reveal a novel role for Rab11a in the IAV life cycle, which could have significant implications for within-host spread, genome reassortment and immune evasion.
甲型流感病毒[IAV]基因组由八个负链 RNA 组成，这些 RNA 以病毒核糖核蛋白[vRNP]的形式包装到病毒粒子中。Rab11a 在 vRNP 通过微管从细胞核转运到质膜的过程中起着至关重要的作用，允许组装和病毒产生。在这里，我们使用隧道纳米管[TNTs]作为分子高速公路，发现 Rab11a 在 IAV vRNP 的细胞间运输中的一个新功能。TNTs 是富含 F-肌动蛋白的小管，将附近细胞的细胞质连接起来。在 IAV 感染的细胞中，Rab11a 与 vRNP 一起被可视化在这些富含肌动蛋白的细胞间连接中。为了更好地研究通过 TNTs 的病毒传播，我们设计了一种感染系统，其中常规的、病毒介导的传播是不可能的。也就是说，我们生成了 HA 缺失的报告病毒，这些病毒不能产生后代病毒，但它们的基因组可以被复制和运输。在这个系统中，观察到 vRNP 向邻近细胞的转移，并且发现这种转移依赖于肌动蛋白和 Rab11a。通过 TNT 转移产生感染性病毒是通过用 HA 缺失病毒感染供体细胞和稳定表达 HA 蛋白的受体细胞来确认的。用感染了遗传上不同的 IAV 的供体细胞混合，进一步显示了 Rab11a 和 TNTs 作为基因组混合和重配的途径在 IAV 感染中的潜力。这些数据因此揭示了 Rab11a 在 IAV 生命周期中的一个新作用，这可能对宿主内传播、基因组重配和免疫逃避有重大影响。
