Background

The COVID-19 pandemic caused by SARS-CoV-2 still rages around the world, threatening public health and the global economy (Wang et al., 2020). Several reverse genetic systems have been reported for generating SARS-CoV-2 replicons and recombinant virus for basic virology research and antiviral development (Hou et al., 2020; Xie et al., 2020; Zhang et al., 2021a and 2021b). However, as SARS-CoV-2 is classified as BSL-3 pathogen, experiments involving authentic virus are restricted to BSL-3 laboratories, which hinders basic research and antiviral discovery. Pseudotyped virus and replicon systems of SARS-CoV-2 that can be used in BSL-2 laboratories have been developed but are limited to studying viral entry and replication, respectively (Nie et al., 2020; Zhang et al., 2021b). Therefore, a cell culture model for SARS-CoV-2 that could safely recapitulate the entire viral life cycle in a BSL-2 facility is urgently needed (Rome and Avorn, 2020).

Recently, we developed such a system in which transcription- and replication-component SARS-CoV-2 virus-like particles (SARS-CoV-2 trVLPs) can be generated (Ju et al., 2021). We replaced the N gene in the SARS-CoV-2 genome with a GFP reporter gene and then provided N in trans in Caco-2 cells ectopically expressing this gene (Caco-2-N). Transducing the Caco-2-N cells with the SARS-CoV-2 GFP/ΔN genome thus allows for the production of trVLPs, which can complete the entire life cycle exclusively in the Caco-2-N packaging cells. In normal cells, these trVLPs can only complete a single-round infection as the packaged viral genome lacks the N gene, which is critical for viral particle assembly. Thus, this system can be safely utilized in BSL-2 level laboratories for SARS-CoV-2 research. In addition, the GFP reporter provides a convenient surrogate readout for virus infection, which facilitates the use of this system for neutralizing antibody determination and high-throughput antiviral screening.

Genetic manipulation of this SARS-CoV-2 trVLPs system is not trivial due to the large size of the viral genome (~30 kb) and presence of multiple toxic elements (Almazan et al., 2014; Xie et al., 2021). Herein, we describe technical details of our cell culture system for production of trVLPs. Overall, the engineering process includes three parts: packaging cell line construction, viral genome-length RNA preparation, and trVLP recovery. Using lentiviral transduction, SARS-CoV-2 N protein can be stably expressed in Caco-2 cells to generate the packaging cell line necessary for trVLP production. Besides, an intein-mediated protein trans-splicing approach (Stevens et al., 2017) was utilized to minimize the chance of N gene recombination into the genome of trVLP. The genome-length viral RNA is prepared by in vitro transcription of a full-length cDNA template generated by in vitro ligation. Briefly, four fragments (A-B, C, D, and E) were designed to cover the entire genome of SARS-CoV-2 GFP/ΔN. Each fragment can be chemically synthesized and then amplified using PCR (Figure 1). After PCR amplification, the fragments, which are flanked by a type IIS restriction enzyme recognition site (BsaI), are digested and then ligated in vitro to assemble the full-length cDNA of the viral genome. As type IIS restriction enzymes recognize asymmetric DNA sequences and cleave at a defined distance outside of their recognition sequence, the fragments are unidirectionally assembled into the full-length cDNA. A T7 promoter and a poly(A) tail were engineered upstream of fragment A and downstream of fragment E, respectively, ultimately allowing for in vitro transcription to produce capped, polyadenylated viral RNA. This genome-length RNA can then be electroporated into the Caco-2-N packaging cell line and trVLPs subsequently collected from the supernatant. Then the trVLPs are amplified and titrated using a tissue-culture infectious dose 50% (TCID₅₀) endpoint dilution assay following the Reed & Muench method (Lindenbach, 2009), and GFP expression is the proxy of trVLP infection. SARS-CoV-2 trVLP can be used for evaluating antivirals and neutralizing antibodies.

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.