Optimized production and fluorescent labeling of SARS-CoV-2 virus-like particles

• Published in: Scientific reports 2022-08, Vol.12 (1), p.14651-14651, Article 14651
• Main Authors: Gourdelier, Manon, Swain, Jitendriya, Arone, Coline, Mouttou, Anita, Bracquemond, David, Merida, Peggy, Saffarian, Saveez, Lyonnais, Sébastien, Favard, Cyril, Muriaux, Delphine
• Format: Article
• Language: English
• Subjects: 631/1647; 631/1647/2204; 631/1647/328; 631/1647/338; 631/326; 631/326/596; 631/337; 631/57; 631/61; 631/61/185; 631/61/350; 631/61/350/1056; Atomic force microscopy; Biotechnology; Cell fusion; COVID-19; Fluorescence; Green fluorescent protein; Humanities and Social Sciences; Internalization; Life cycles; Life Sciences; Microbiology and Parasitology; multidisciplinary; Pandemics; Proteins; RNA viruses; Science; Science (multidisciplinary); Severe acute respiratory syndrome coronavirus 2; Structural proteins; Virology; Virus-like particles; Western blotting
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-022-18681-z

SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemic that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100 nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the viral RNA ratio in infected cells. This allows SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and mature S. Furthermore, fluorescent or photoconvertible VLPs were generated by adding a fluorescent protein tag on N or M mixing with unlabeled viral proteins and characterized by western blots, atomic force microscopy coupled to fluorescence and immuno-spotting. Thanks to live fluorescence and super-resolution microscopies, we quantified VLPs size and concentration. SARS-CoV-2 VLPs present a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a concentration of 10e12 VLP/ml. In this condition, we were able to establish the incorporation of the Spike in the fluorescent VLPs. Finally, the Spike functionality was assessed by monitoring fluorescent MNES-VLPs docking and internalization in human pulmonary cells expressing or not the receptor hACE2. Results show a preferential maturation of S on N(GFP) labeled VLPs and an hACE2-dependent VLP internalization and a potential fusion in host cells. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.