Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.
Highlights
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an ongoing pandemic[1]
Nucleocapsid translated from a subgenomic RNA of SARS-CoV-2 has multiple functions and its primary function is participation in genomic RNA package and virus particle release
To test whether the function of N could be complemented in trans, we constructed SARS-CoV-2 GFP/ΔN genome, in which we replaced the regions encoding viral N based on MN908947 genome with GFP reporter gene, and Caco-2 cells, an immortalized cell line of human colorectal adenocarcinoma cells, as packaging cell lines which stably express viral N protein by lentiviral transduction (Fig 1)
Summary
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an ongoing pandemic[1]. The genome encodes an RNAdependent RNA-polymerase complex (nsp, nsp and nsp12), RNA capping machinery (nsp, nsp, nsp and 16) and additional enzymes such as proteases (the nsp PLpro and the nsp5 3CLpro) which cleave viral polyproteins[4,5]. The primary function of N protein is to package the *30 kb single stranded, 50-capped positive-strand viral genome RNA into a ribonucleoprotein (RNP) complex. The accessory proteins, encoded by ORF3a, ORF6, ORF7a, ORF7b, and ORF8 genes, are not directly involved in viral replication but interfere with the host innate immune response or are of unknown function[3,8,9]
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