Broich Lukas, Wullenkord Hannah, Osman Maria Kaukab, Fu Yang, Müsken Mathias, Reuther Peter, Brönstrup Mark, Sieben Christian
Nanoscale Infection Biology Group, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Nat Commun. 2025 Apr 25;16(1):3846. doi: 10.1038/s41467-025-58935-8.
During infection, individual virions trigger specific cellular signaling at the virus-cell interface, a nanoscale region of the plasma membrane in direct contact with the virus. However, virus-induced receptor recruitment and cellular activation are transient processes that occur within minutes at the nanoscale. Hence, the temporal and spatial kinetics of such early events often remain poorly understood due to technical limitations. To address this challenge, we develop a protocol to covalently immobilize labelled influenza A viruses on glass surfaces before exposing them to live epithelial cells. Our method extends the observation time for virus-plasma membrane association while minimizing viral modifications, facilitating live imaging of virus-cell interactions. Using single-molecule super-resolution microscopy, we investigate virus-receptor interaction showing that viral receptors exhibit reduced mobility at the virus-binding site, which leads to a specific local receptor accumulation and turnover. We further follow the dynamics of clathrin-mediated endocytosis at the single-virus level and demonstrate the recruitment of adaptor protein 2 (AP-2), previously thought to be uninvolved in influenza A virus infection. Finally, we examine the nanoscale organization of the actin cytoskeleton at the virus-binding site, showing a local and dynamic response of the cellular actin cortex to the infecting virus.
在感染过程中，单个病毒粒子在病毒 - 细胞界面触发特定的细胞信号传导，该界面是质膜中与病毒直接接触的纳米级区域。然而，病毒诱导的受体募集和细胞激活是在纳米尺度上几分钟内发生的短暂过程。因此，由于技术限制，此类早期事件的时空动力学往往仍知之甚少。为应对这一挑战，我们开发了一种方案，在将标记的甲型流感病毒暴露于活上皮细胞之前，将其共价固定在玻璃表面。我们的方法延长了病毒 - 质膜结合的观察时间，同时最大限度地减少病毒修饰，便于对病毒 - 细胞相互作用进行实时成像。使用单分子超分辨率显微镜，我们研究病毒 - 受体相互作用，结果表明病毒受体在病毒结合位点的流动性降低，这导致特定的局部受体积累和周转。我们进一步在单病毒水平跟踪网格蛋白介导的内吞作用的动力学，并证明衔接蛋白2（AP - 2）的募集，此前认为其不参与甲型流感病毒感染。最后，我们研究了病毒结合位点处肌动蛋白细胞骨架的纳米级组织，显示细胞肌动蛋白皮层对感染病毒的局部和动态反应。
