DNA transfection

HEK293T cells were seeded into a 6-well plate or 10 mL dishes 24 h before transfection. Plasmids expressing viral proteins were transfected with CaCl₂/HBS2X (50 mM HEPES pH 7.1, 280 mM NaCl, 1.5 mM Na₂HPO₄) (v/v) into HEK293T cells. A total of 1.4 µg of mixed plasmids was applied for the transfection of a well (for 6-well plate) and 0.7 µg/well for individual plasmid. Co-transfection of double, triple or quadruple plasmids was conducted with M, N, E and S with plasmid ratio 3:3:3:5 respectively (0.3 µg:0.3 µg:0.3 µg:0.5 µg per well to 6-well plate for example) in the first assay as shown in Fig. 1B. Optimization of SARS-CoV-2 VLPs was performed by co-transfecting M, N, E and S with a plasmid ratio of 3:12:2:5 respectively corresponding to the molecular viral RNA ratio found in infected cells^17,18, corresponding, for an example, to a total of 2,2 µg/well of 6 well plate (with M:N:E:S corresponding to 0.3 µg:1.2 µg:0.2 µg:0.5 µg of transfected plasmid) as shown in Fig. 1C. Optimization of fluorescent SARS-CoV-2 VLPs production was performed by co-transfecting M, M(GFP) or M(mCherry), N, E and S with plasmid ratio of 2:2:12:2:5 respectively, and M, N, N(GFP) or N(mEOS2), E and S with plasmid ratio of 3:9:3:2:5 respectively. Then, transfected cells were washed 6 h later with phosphate-buffered saline (PBS 1x) and harvested 24 h to 48 h post-transfection with Exofree medium. Attached cells were washed with PBS, harvested, centrifuged at 1500 rpm for 5 min at 4 °C and lysed with RIPA lysis buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (ThermoFisher) supplemented with protease inhibitor cocktail (Sigma). Following centrifugation at 15,000 rpm for 10 min at 4 °C, cell lysates were collected. The protein concentrations in cell lysates were estimated using Bradford assays. Under each condition, the percentage of cell viability was measured by trypan blue cell counting before cell lysis.

Optimization of SARS-CoV-2 VLPs assembly and particle analysis using immunoblots and atomic force microscopy imaging. (A) Scheme of VLPs production and purification. Thanks to BioRender for images. (B) Western blot of cell lysates and VLPs of HEK293T cells transfected with M, N, E or S as indicated for each lane. The transfected M/N/E/S plasmid ratios are respectively MNES 3:3:3:5; MNE 3:3:3; MNS: 3:3:5, NES 3:3:5, MES 3:3:5, ME 3:3, MS 3:5, M 3, N 3, E 3, S 5. N = 1. (C) Western blot of cell lysates and VLPs of HEK293T cells transfected with the indicated 3:3:3: ± 5 and 3:12:2: ± 5 M, N, E ± S transfected plasmid ratios. Right panel: Graph representing the percentage of M, N, E and S in released VLPs after quantification of the western blot band intensities for each condition. N = 3 independent experiments. (D) AFM imaging of SARS-CoV-2 MNES VLPs and MNE VLPs using quantitative QI mode AFM in TNE buffer. For each condition, a topographic image of 2.5 µm × 2.5 µm and zoom of a particle is shown, with a 3D projection. The color gradient for the Z scale is the same for all panels. The topographical profile plots along the white line are shown for each VLP. N = 2 with 18 VLP analyzed.