A Nucleocapsid-based Transcomplementation Cell Culture System of SARS-CoV-2 to Recapitulate the Complete Viral Life Cycle

Abstract

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As this virus is classified as a biosafety level-3 (BSL-3) agent, the development of countermeasures and basic research methods is logistically difficult. Recently, using reverse genetics, we developed a BSL-2 cell culture system for production of transcription- and replication-component virus-like-particles (trVLPs) by genetic transcomplementation. The system consists of two parts: SARS-CoV-2 GFP/ΔN genomic RNA, in which the nucleocapsid (N) gene, a critical gene for virion packaging, is replaced by a GFP reporter gene; and a packaging cell line for ectopic expression of N (Caco-2-N). The complete viral life cycle can be recapitulated and confined to Caco-2-N cells, with GFP positivity serving as a surrogate readout for viral infection. In addition, we utilized an intein-mediated protein splicing technique to split the N gene into two independent vectors and generated the Caco-2-N^intein cells as a packaging cell line to further enhance the security of this cell culture model. Altogether, this system provides for a safe and convenient method to produce trVLPs in BSL-2 laboratories. These trVLPs can be modified to incorporate desired mutations, permitting high-throughput screening of antiviral compounds and evaluation of neutralizing antibodies. This protocol describes the details of the trVLP cell culture model to make SARS-CoV-2 research more readily accessible.

Keywords: BSL-2; Nucleocapsid; Reverse genetics; SARS-CoV-2; Transcomplementation; trVLP.

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-N^Intein cells. trVLPs can infect and replicate in Caco-2-N or Caco-2-N^Intein 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.

Figure 2.. Agarose gel electrophoresis verification of DNA and RNA fragments for trLVPs generation.

(A) The genome sequence of trVLPs was divided into four fragments and each fragment PCR amplified. The purified PCR products of each fragment were determined by agarose gel. (B) PCR products of C-D-E and purified ligation products of full-length DNA (FL DNA). (C) Agarose gel analysis of SARS-CoV-2 N gene PCR products. (D) Agarose gel analysis of viral full-length RNA (FL-RNA) and (E) N gene mRNA generated by in vitro transcription using the FL DNA genome and N gene PCR products, respectively, as template. The black arrow indicates the FL RNA and N RNA, respectively.

Figure 3.. Fluorescence microscopy analysis of Caco-2-N cells infected with SARS-CoV-2 GFP/ΔN.

(A) GFP and bright-field images of Caco-2-N cells at 12 h post electroporation. (B) Supernatants of Caco-2-N cells at 24 h post electroporation were collected and used to infect naive Caco-2-N cells in a 24-well plate. The GFP and bright-field images 24 h and 48 h post infection are shown.

Figure 4.. Analysis by RT-PCR of the GFP gene in trLVPs.

RT-PCR validation of the GFP gene in SARS-CoV-2 GFP/ΔN trVLPs generated in Caco-2-N cells. The expected DNA size (A) and agarose electrophoresis analysis of the PCR products (B) are shown.