Université Paris-Saclay, Université de Versailles St. Quentin, UMR 1173 (2I), INSERM, Versailles, France.
Université Paris-Saclay, Université de Versailles St. Quentin; UMR 1173 (2I), INSERM; Assistance Publique des Hôpitaux de Paris, Hôpital Ambroise Paré, Laboratoire de Microbiologie, DMU15; Versailles, France.
PLoS Pathog. 2022 Jul 7;18(7):e1010619. doi: 10.1371/journal.ppat.1010619. eCollection 2022 Jul.
Respiratory syncytial virus (RSV) is the primary cause of severe respiratory infection in infants worldwide. Replication of RSV genomic RNA occurs in cytoplasmic inclusions generating viral ribonucleoprotein complexes (vRNPs). vRNPs then reach assembly and budding sites at the plasma membrane. However, mechanisms ensuring vRNPs transportation are unknown. We generated a recombinant RSV harboring fluorescent RNPs allowing us to visualize moving vRNPs in living infected cells and developed an automated imaging pipeline to characterize the movements of vRNPs at a high throughput. Automatic tracking of vRNPs revealed that around 10% of the RNPs exhibit fast and directed motion compatible with transport along the microtubules. Visualization of vRNPs moving along labeled microtubules and restriction of their movements by microtubule depolymerization further support microtubules involvement in vRNPs trafficking. Approximately 30% of vRNPs colocalize with Rab11a protein, a marker of the endosome recycling (ER) pathway and we observed vRNPs and Rab11-labeled vesicles moving together. Transient inhibition of Rab11a expression significantly reduces vRNPs movements demonstrating Rab11 involvement in RNPs trafficking. Finally, Rab11a is specifically immunoprecipitated with vRNPs in infected cells suggesting an interaction between Rab11 and the vRNPs. Altogether, our results strongly suggest that RSV RNPs move on microtubules by hijacking the ER pathway.
呼吸道合胞病毒（RSV）是全球婴儿严重呼吸道感染的主要原因。RSV 基因组 RNA 的复制发生在细胞质包含体中，产生病毒核糖核蛋白复合物（vRNPs）。然后，vRNPs 到达质膜的组装和出芽部位。然而，确保 vRNPs 运输的机制尚不清楚。我们生成了一种携带荧光 RNP 的重组 RSV，使我们能够在活感染细胞中可视化移动的 vRNPs，并开发了一种自动成像管道，以高通量表征 vRNPs 的运动。vRNPs 的自动跟踪显示，约 10%的 RNP 表现出快速和定向运动，与沿微管的运输兼容。可视化沿标记的微管移动的 vRNPs 以及微管解聚对其运动的限制进一步支持微管参与 vRNPs 运输。约 30%的 vRNPs 与 Rab11a 蛋白共定位，Rab11a 是内体再循环（ER）途径的标志物，我们观察到 vRNPs 和 Rab11 标记的囊泡一起移动。Rab11a 表达的瞬时抑制显著减少了 vRNPs 的运动，表明 Rab11 参与了 RNP 的运输。最后，Rab11a 可在感染细胞中与 vRNPs 特异性免疫沉淀，表明 Rab11 与 vRNPs 之间存在相互作用。总之，我们的研究结果强烈表明，RSV RNP 通过劫持 ER 途径在微管上移动。
