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

Manon Gourdelier (1) , Jitendriya Swain (1) , Coline Arone (1) , Anita Mouttou (1) , David Bracquemond (1) , Peggy Merida (1) , Saveez Saffarian (2) , Sébastien Lyonnais (3) , Cyril Favard (1) , Delphine Muriaux (1, 3)

1 IRIM - Institut de Recherche en Infectiologie de Montpellier
2 University of Utah
3 CEMIPAI - Centre d’études des Maladies Infectieuses et Pharmacologie Anti-Infectieuse - [Montpellier]

Abstract 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.

Domaines: Virologie; Biotechnologies

Manon Gourdelier, Jitendriya Swain, Coline Arone, Anita Mouttou, David Bracquemond, et al.. Optimized production and fluorescent labeling of SARS-CoV-2 virus-like particles. Scientific Reports, 2022, 12 (1), pp.14651. ⟨10.1038/s41598-022-18681-z⟩. ⟨hal-03875922⟩