Single-particle quantification of SARS-CoV-2 virus-like particles using flow virometry

SARS-CoV-2 virus-like particles (VLPs) were generated by co-transfecting HEK 293T cells with four plasmids, two for expression of the spike (S) or nucleocapsid (N) protein with a 3xHA tag at the C-terminus, one for expression of the membrane (M) protein, and one for expression of the envelope (E) protein. Co-localization of the S and N proteins within the VLPs was confirmed by Western blot analysis using anti-HA antibodies. To enable fluorescent quantification of VLPs, the C-terminus of the S protein was fused to enhanced green fluorescent protein (EGFP), and the N protein was tagged with EGFP at either the N- or C-terminus. Transient transfection of 293T cells with (S-GFP)NME, S(N-GFP)ME, or S(GFP-N)ME plasmids efficiently produced fluorescent VLPs, each demonstrating the ability to enter A549-hACE2 cells. In addition, a two-plasmid system was developed to simplify fluorescent VLP production by incorporating internal ribosomal entry site elements between N3xHA and S-GFP and between the M and E genes. Transfection of 293T cells with these plasmids produced VLPs with a fourfold higher N protein concentration (800 ng/mL) compared to the four-plasmid system (200 ng/mL), as measured by ELISA. Flow virometry analysis confirmed production of VLPs with an average diameter of 80 nm and a concentration of 1.68 × 10⁸ particles/ml. This study demonstrates that production of fluorescent VLPs using a two-plasmid system is more efficient than the traditional four-plasmid approach. We also demonstrate that flow virometry is a robust method for single-particle analysis of fluorescent VLPs for determining their size and concentration.