Figure 1. Overview of production of SARS-CoV-2 GFP/ΔN trVLPs.
The N gene of SARS-CoV-2 was replaced with the GFP gene, and the cDNA genome divided into four fragments designated as A-B, C, D, and E. Each of these fragments was chemically synthesized and then PCR amplified and assembled by restriction enzyme digestion and in vitro ligation to create the full-length cDNA. The full-length RNA genome was generated by in vitro transcription of the full-length cDNA. This RNA genome can then be electroporated into the packaging cell line, Caco-2-N, to produce trVLPs. At 24 h post electroporation, the supernatant of electroporated cells is collected and can be used to inoculate Caco-2, Caco-2-N, or Caco-2-NIntein cells. trVLPs can infect and replicate in Caco-2-N or Caco-2-NIntein cells and can be secreted into the supernatant. However, trVLPs only complete a single-round infection in Caco-2 cells due to the absence of viral N protein.