Ma Yingxin, Mao Guobin, Wu Guoqiang, Chen Minghai, Qin Fujun, Zheng Luping, Zhang Xian-En
CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
National Key Laboratory of Biomacromolecules, CAS Center for Biological Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
ACS Appl Mater Interfaces. 2021 Jun 2;13(21):24477-24486. doi: 10.1021/acsami.1c03897. Epub 2021 May 7.
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.
伪病毒策略使高致病性病毒的研究在不受高级别生物安全设施限制的情况下成为可能，从而极大地促进了病毒学发展，并被用于严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的研究。在此，我们利用人类免疫缺陷病毒1伪病毒生产系统和SARS-CoV-2刺突（S）糖蛋白构建了一种双色伪SARS-CoV-2病毒，其包膜用亲脂性染料（DiO）标记，病毒核心的基因组RNA相关病毒蛋白R（Vpr）与mCherry融合。通过这种双色标记策略，不仅可以追踪整个病毒的运动，还可以追踪标记成分的去向。将伪病毒颗粒应用于在四种人类呼吸道细胞中以单颗粒水平追踪病毒进入：鼻上皮细胞（HNEpC）、肺泡上皮细胞（HPAEpiC）、支气管上皮细胞（BEP-2D）和口腔上皮细胞（HOEC）。伪SARS-CoV-2进入宿主细胞并将病毒核心释放到细胞质中，这清楚地表明宿主进入主要通过内吞作用发生。发现感染效率与宿主细胞表面SARS-CoV-2已知受体血管紧张素转换酶2（ACE2）的表达相关。我们相信，本研究中创建的双色荧光标记伪病毒系统可作为一种有用工具，用于SARS-CoV-2/2019冠状病毒病的多种用途。
