biostudies-literatureZhang YNational Natural Science Foundation of China2001031https://www.ebi.ac.uk/biostudies/studies/S-EPMC7883248biostudies-literatureUnknown5(2)The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed. In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.Small methodsVirus-Free and Live-Cell Visualizing SARS-CoV-2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors.PMC7883248U1905205819020578199314904181871316Liu LZhang JCao JShen CZhang LWang JYu HHou WShi YGe SWei MCheng TWang SXia NChen YWu YYuan QYuan LZheng QYang CXu JFu BLi ZZhang TNian SHu JZhang YMa JYang TYe JXiong HfalseVirus-Free and Live-Cell Visualizing SARS-CoV-2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors.The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed. In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.2021-01-01T00:00:00Z2021 Feb2026-05-04T18:29:09.822Z2021-02-21T10:47:40ZS-EPMC78832483361490710.1002/smtd.202001031