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

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.

Development of Fluorescence-Tagged SARS-CoV-2 Virus-like Particles by a Tri-Cistronic Vector Expression System for Investigating the Cellular Entry of SARS-CoV-2

“…In SARS-CoV-2 studies, the real virus or infectious materials must be manipulated in biosafety level 3 laboratories, limiting the investigation rate of the SARS-CoV-2 life cycle. The application of substitutes such as pseudovirus, viral protein-expressing cells and VLPs have been developed for SARS-CoV-2 studying [25]. The pseudovirus of SARS-CoV-2 was a kind of chimeric virus with SARS-CoV-2 spike and other structure proteins derived from different viruses, such as lentivirus or Vesicular stomatitis virus (VSV) [26,27].…”

“…The labeling of SARS-CoV-2 VLPs with fluorescence could be very useful to visualize either viral entry or assembly in target cells. The fusion of structural proteins of SARS-CoV-2 (including S, M and N proteins) with a fluorescence protein such as green fluorescence protein (GFP) had been used to visualize SARS-CoV-2 VLPs in cells [13,25]. Given that the molecular weight of fluorescent proteins is around 25-30 kDa, it is possible that tagging with fluorescence proteins may hinder the biological functions of SARS-CoV-2 structure proteins.…”

Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions

“…It has been shown that co-transfection of plasmids expressing each of the four SARS-CoV-2 structural proteins allows for secretion of virus-like particles (VLPs) [19,61,62], which is concordant with earlier studies with other CoV structural proteins [63,64]. However, we observed that when Spike, E, M and N expression vectors were co-transfected into cells, 6-TG treatment not only altered the accumulation and processing of Spike, but also dramatically reduced accumulation of E and M proteins (Fig 5A).…”

Optimization of resonant dielectric multilayer for enhanced fluorescence imaging

“…More recently, TIRF-M was indeed combined with super-critical angle fluorescence detection for high axial resolution and sharp localization pointing precision [5]. In the field of virology, these techniques have proven their strength and use [6][7][8][9][10][11]. For the lack of sensitivity, as the fluorescence emission is, among others, proportional to the intensity of the excitation field, we proposed to act on the local evanescent exciting field by introducing resonant dielectric multilayer (DM) coated coverslips while limiting its impact over the fluorescence collection efficiency.…”