Dynamic Dissection of the Endocytosis of Porcine Epidemic Diarrhea Coronavirus Cooperatively Mediated by Clathrin and Caveolae as Visualized by Single-Virus Tracking.
Dynamic Dissection of the Endocytosis of Porcine Epidemic Diarrhea Coronavirus Cooperatively Mediated by Clathrin and Caveolae as Visualized by Single-Virus Tracking.
复制标题
单病毒追踪可视化网格蛋白和小窝协同介导的猪流行性腹泻冠状病毒内吞作用的动态解析
作者：
Li Y;Wang J;Hou W;Shan Y;Wang S;Liu F
新出现和再次出现的冠状病毒在世界范围内导致严重的人类和动物流行病。对于包括冠状病毒在内的许多包膜病毒来说，打破质膜屏障显然是一个关键而复杂的过程，其中包含多个动态步骤。摘要冠状病毒(CoV)已引起人类和动物的严重疾病。胞内途径，如网状蛋白介导的内吞作用(CME)和小窝介导的内吞作用(CavME)，在冠状病毒穿透细胞膜屏障中起着重要作用。在本研究中，揭示了一种新型的冠状病毒进入方式，在这种方式下，网状蛋白和小窝可以协同介导猪流行性腹泻冠状病毒(PEDV)的内吞作用。利用多色活细胞成像技术，对荧光标记的网状蛋白结构、小窝结构和PEDV进行了动态观察。在病毒空泡形成过程中，我们发现在细胞膜附近有网状结构与小凹融合，病毒穿透细胞膜的平均时间在∼3 内，表现出快速进入细胞膜的过程。此外，根据分子筛和小窝结构的动态募集和病毒的运动性，直接证据还表明，大约20%的PEDV可以通过CME和CavME流产进入。此外，还直接解剖了PEDV从网状结构和小凹结构到早期内体、从早期内体到晚期内体的动态转运以及病毒的融合，在∼6.8min内，PEDV的融合主要发生在PEDV到晚期内体的6.8min内。总体而言，这项工作系统地揭开了PEDV感染的早期步骤，扩大了我们对CoV感染机制的理解。重要性新出现和重新出现的冠状病毒在世界范围内造成严重的人类和动物流行病。对于包括冠状病毒在内的许多包膜病毒来说，打破质膜屏障显然是一个关键而复杂的过程，其中包含多个动态步骤。虽然人们对冠状病毒内吞途径的机制已经有了很大的了解，但还没有实现对个体猪流行性腹泻冠状病毒(PEDV)内化的直接实时成像。在这项研究中，我们不仅剖析了PEDV通过网状蛋白介导的内吞作用和小窝介导的内吞作用进入PEDV的动力学以及内小体转运和病毒融合的动力学，而且还发现了一种新的有效的冠状病毒进入方式，在这种方式下，网状蛋白和小窝可以协同地介导PEDV的内吞作用。此外，我们还发现了PEDV流产内吞作用的存在。综上所述，PEDV通过网状蛋白和小窝结构的协同进入和PEDV流产的内吞作用为冠状病毒穿透质膜屏障提供了新的见解。
Emerging and re-emerging coronaviruses cause serious human and animal epidemics worldwide. For many enveloped viruses, including coronavirus, it is evident that breaking the plasma membrane barrier is a pivotal and complex process, which contains multiple dynamic steps. ABSTRACT Coronaviruses (CoVs) have caused severe diseases in humans and animals. Endocytic pathways, such as clathrin-mediated endocytosis (CME) and caveolae-mediated endocytosis (CavME), play an important role for CoVs to penetrate the cell membrane barrier. In this study, a novel CoV entry manner is unraveled in which clathrin and caveolae can cooperatively mediate endocytosis of porcine epidemic diarrhea coronavirus (PEDV). Using multicolor live-cell imaging, the dynamics of the fluorescently labeled clathrin structures, caveolae structures, and PEDV were dissected. During CavME of PEDV, we found that clathrin structures can fuse with caveolae near the cell plasma membrane, and the average time of PEDV penetrating the cell membrane was within ∼3 min, exhibiting a rapid course of PEDV entry. Moreover, based on the dynamic recruitment of clathrin and caveolae structures and viral motility, the direct evidence also shows that about 20% of PEDVs can undergo an abortive entry via CME and CavME. Additionally, the dynamic trafficking of PEDV from clathrin and caveolae structures to early endosomes, and from early endosomes to late endosomes, and viral fusion were directly dissected, and PEDV fusion mainly occurred in late endosomes within ∼6.8 min after the transport of PEDV to late endosomes. Collectively, this work systematically unravels the early steps of PEDV infection, which expands our understanding of the mechanism of CoV infection. IMPORTANCE Emerging and re-emerging coronaviruses cause serious human and animal epidemics worldwide. For many enveloped viruses, including coronavirus, it is evident that breaking the plasma membrane barrier is a pivotal and complex process, which contains multiple dynamic steps. Although great efforts have been made to understand the mechanisms of coronavirus endocytic pathways, the direct real-time imaging of individual porcine epidemic diarrhea coronavirus (PEDV) internalization has not been achieved yet. In this study, we not only dissected the kinetics of PEDV entry via clathrin-mediated endocytosis and caveolae-mediated endocytosis and the kinetics of endosome trafficking and viral fusion but also found a novel productive coronavirus entry manner in which clathrin and caveolae can cooperatively mediate endocytosis of PEDV. Moreover, we uncovered the existence of PEDV abortive endocytosis. In summary, the productive PEDV entry via the cooperation between clathrin and caveolae structures and the abortive endocytosis of PEDV provide new insights into coronavirus penetrating the plasma membrane barrier.