病毒内化到靶细胞中的效率是感染性的主要决定因素。SARS-CoV-2内化通过S蛋白介导的细胞结合发生,然后与质膜直接融合或内吞作用,然后与内体膜融合。尽管病毒内化的关键作用,所涉及的过程的精确动力学仍然难以捉摸。我们开发了一条管道,结合了活细胞显微镜和先进的图像分析,用于测量单个SARS-CoV-2-病毒样颗粒(VLP)的多个内化相关分子事件的速率,包括内体侵入和pH变化。我们的活细胞成像实验表明,在与质膜结合后几分钟,VLP通过动力蛋白依赖性断裂进入RAP5阴性内体。不到两分钟后,VLP速度增加与pH下降到5以下平行,但这两个事件并不相关。通过荧光标记的核衣壳蛋白共成像,我们表明核衣壳释放的动力学与VLP酸化相似。Omicron突变或S蛋白多碱基切割位点的废除都不会影响VLP内化的速率,表明它们在此过程中不赋予任何显著的优点或缺点。最后,我们观察到VLP内化在VeroE6中发生的速度比A549细胞快两到三倍,这可能有助于前细胞系对SARS-CoV-2感染的更大敏感性。一起来看,我们对VLP内化相关过程动力学的精确测量揭示了它们对SARS-CoV-2在细胞中传播有效性的贡献.
The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression and pH change. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into RAP5-negative endosomes via dynamin-dependent scission. Less than two minutes later, VLP speed increases in parallel with a pH drop below 5, yet these two events are not interrelated. By co-imaging fluorescently labeled nucleocapsid proteins, we show that nucleocapsid release occurs with similar kinetics to VLP acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site affected the rate of VLP internalization, indicating that they do not confer any significant advantages or disadvantages during this process. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells.