Dual-Fluorescence Labeling Pseudovirus for Real-Time Imaging of Single SARS-CoV-2 Entry in Respiratory Epithelial Cells.
The pseudovirus strategy makes studies of highly pathogenic viruses feasible without the restriction of high-level biosafety facility, thus greatly contributing to virology and is used in the research studies of SARS-CoV-2. Here, we generated a dual-color pseudo-SARS-CoV-2 virus using a human immunodeficiency virus-1 pseudovirus production system and the SARS-CoV-2 spike (S) glycoprotein, of which the membrane was labeled with a lipophilic dye (DiO) and the genomic RNA-related viral protein R (Vpr) of the viral core was fused with mCherry. With this dual-color labeling strategy, not only the movement of the whole virus but also the fate of the labeled components can be traced. The pseudovirions were applied to track the viral entry at a single-particle level in four types of the human respiratory cells: nasal epithelial cells (HNEpC), pulmonary alveolar epithelial cells (HPAEpiC), bronchial epithelial cells (BEP-2D), and oral epithelial cells (HOEC). Pseudo-SARS-CoV-2 entered into the host cell and released the viral core into the cytoplasm, which clearly indicates that the host entry mainly occurred through endocytosis. The infection efficiency was found to be correlated with the expression of the known receptor of SARS-CoV-2, angiotensin-converting 2 (ACE2) on the host cell surface. We believe that the dual-color fluorescently labeled pseudovirus system created in this study can be applied as a useful tool for many purposes in SARS-CoV-2/COVID-19.
['Ma|Yingxin|Y|http://orcid.org/0000-0002-2899-6597', 'Mao|Guobin|G|http://orcid.org/0000-0002-7665-4129', 'Wu|Guoqiang|G|', 'Chen|Minghai|M|', 'Qin|Fujun|F|', 'Zheng|Luping|L|', 'Zhang|Xian-En|XE|http://orcid.org/0000-0003-1347-3168']
D000085962:Angiotensin-Converting Enzyme 2 D004705:Endocytosis D004847:Epithelial Cells D005453:Fluorescence D005456:Fluorescent Dyes D057809:HEK293 Cells D015497:HIV-1 D006801:Humans D009297:Nasal Mucosa D011650:Pulmonary Alveoli D000086402:SARS-CoV-2 D064370:Spike Glycoprotein, Coronavirus D053586:Virus Internalization
respiratory epithelial cells pseudo-SARS-CoV-2 virus dual fluorescence endocytic entry single-virus imaging