SARS-CoV-2入胞活细胞示踪平台的建立

Establishment of A Live-cell Tracking Platform for SARS-CoV-2 Entry

新型冠状病毒(SARS-CoV-2)由于其高传染性已造成全球大流行。目前关于SARS-CoV-2依赖内吞途径进入细胞的研究偏向于药物抑制实验或固定样品的蛋白共定位实验,而对于其入胞时与细胞内吞结构相互作用的动力学机制研究较少。本研究首先基于慢病毒系统包装出可在生物安全二级实验室(BSL-2)进行操作的SARS-CoV-2假病毒,之后利用膜染料对假病毒的囊膜进行荧光标记,并进行鉴定。通过免疫荧光方法观察到假病毒与网格蛋白包被的内吞结构共定位,进一步在网格蛋白敲低的Caco-2细胞系上进行假病毒感染,检测到荧光素酶活性显著下降,这些结果确定了网格蛋白介导的内吞作用参与假病毒感染。最后基于单病毒示踪技术,通过共聚焦显微镜对假病毒入胞过程进行活细胞实时拍摄,选取两个具有代表性的假病毒粒子依赖网格途径入胞的典型视野,并对假病毒动力学进行分析。本研究成功建立了SARS-CoV-2假病毒入胞活细胞示踪平台,可应用于研究单个假病毒粒子入胞过程动力学机制。该平台对SARS-CoV-2入胞机制的研究具有重大意义。

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused a global pandemic due to its high infectivity.At present,researches on SARS-CoV-2 entry via endocytic pathways are based on drug inhibition experiments or proteins colocalization experiments with fixed samples.There are few studies on the kinetic mechanism of the interaction between the virus and the endocytic structure during virus entry.In this study,SARS-CoV-2 pseudovirus that can be operated in biosafety level 2 laboratory(BSL-2)was packaged based on a lentiviral system,then the envelope of the pseudovirus was fluorescence labeled with the membrane dye,and the labeled virus was characterized.The colocalization of the pseudovirus and the clathrin-coated endocytic structures were observed by immunofluorescence assay.Pseudovirus infection was further carried out on the Caco-2 cell line with clathrin knock-down,and result showed that the luciferase activity was significantly decreased.These results indicate the involvement of clathrin-mediated endocytosis(CME)in pseudovirus infection.Finally,based on the single-virus tracking technique,live cells were photographed real time during the pseudovirus entry by confocal microscopy.Two representative views of pseudovirus particles entry via CME were selected,and the kinetics was analyzed.This study established a SARS-CoV-2 pseudovirus entry tracking platform for studying the kinetic mechanism of single pseudovirus particle entry.