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

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.

Minimal system for assembly of SARS-CoV-2 virus like particles.

SARS-CoV-2 virus is the causative agent of COVID-19. Here we demonstrate that non-infectious SARS-CoV-2 virus like particles (VLPs) can be assembled by co-expressing the viral proteins S, M and E in mammalian cells. The assembled SARS-CoV-2 VLPs possess S protein spikes on particle exterior, making them ideal for vaccine development. The particles range in shape from spherical to elongated with a characteristic size of 129 ± 32 nm. We further show that SARS-CoV-2 VLPs dried in ambient conditions can retain their structural integrity upon repeated scans with Atomic Force Microscopy up to a peak force of 1 nN.