Abstract
The development of antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hampered by the lack of efficient cell-based replication systems that are amenable to high-throughput screens in biosafety level 2 laboratories. Here we report that stable cell clones harboring autonomously replicating SARS-CoV-2 RNAs without spike (S), membrane (M), and envelope (E) genes can be efficiently derived from the baby hamster kidney (BHK-21) cell line when a pair of mutations were introduced into the non-structural protein 1 (Nsp1) of SARS-CoV-2 to ameliorate cellular toxicity associated with virus replication. In a proof-of-concept experiment we screened a 273-compound library using replicon cells and identified three compounds as novel inhibitors of SARS-CoV-2 replication. Altogether, this work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs. IMPORTANCE SARS-CoV-2 replicon systems that have been reported up to date were unsuccessful in deriving stable cell lines harboring non-cytopathic replicons. The transient expression of viral sgmRNA or a reporter gene makes it impractical for industry-scale screening of large compound libraries using these systems. Here, for the first time, we derived stable cell clones harboring the SARS-CoV-2 replicon. These clones may now be conveniently cultured in a standard BSL-2 laboratory for high throughput screen of compound libraries. Additionally, our stable replicon cells represent a new model system to study SARS-CoV-2 replication.
Keywords: SARS-CoV-2; drug screen; replication; replicon; stable cell clone.
【저자키워드】 SARS-CoV-2, Replication, replicon, drug screen, stable cell clone., 【초록키워드】 coronavirus, Mutation, Antiviral, Genetic, antiviral drugs, Toxicity, severe acute respiratory syndrome Coronavirus, kidney, biosafety, replicon, Viral, non-structural protein, nsp1, membrane, virus replication, SARS-CoV-2 RNA, experiment, respiratory, reporter, SARS-CoV-2 replication, cellular, Analysis, cell lines, sgmRNA, reporter gene, biosafety level, level 2, stable cell, acute respiratory syndrome, acute respiratory syndrome coronavirus, acute respiratory syndrome coronavirus 2, inhibitors of SARS-CoV-2, Compound, BSL-2 laboratory, pair, cell line, stable cell lines, transient expression, clone, Cell, robust, lack, reported, screened, functional, introduced, BHK-21, stable cell line, the SARS-CoV-2, 【제목키워드】 drug, Screen, SARS-CoV-2 RNA, STABLE,