A Convenient and Biosafe Replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery

Abstract

SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all studies on the live virus are strictly confined in the biosafety level 3 (BSL3) laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious. In this study, we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome (BAC) vector with deletion of the spike (S) gene. Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein. Therefore, such a replicon system is not infectious and can be used in ordinary biological laboratories. We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus. By mutational analysis of nsp12 and nsp14, we showed that the RNA polymerase, exonuclease, and cap N7 methyltransferase play essential roles in genome replication and sgRNA production. We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors. Thus, such a one-plasmid system is biosafe and convenient to use, which will benefit both fundamental research and development of antiviral drugs.

Keywords: Antiviral drug screening; Replicon; Reverse genetics; SARS-CoV-2.

Fig. 1

Construction of a SARS-CoV-2 replicon. A Schematic of the genome of SARS-CoV-2 and restriction sites selected for cloning. B The cloning vector for replicon with designed restriction sites. C Schematic of the fragments for cloning with indicated restriction sites, elements for transcription, and positions in the viral genome. D The fragments for cloning were examined in agarose gel electrophoresis. E The organization of the SARS-CoV-2 replicon. Note that SacII and AscI restriction sites were inserted downstream the TRS of S gene which is deleted to eliminate the ability of replicon to generating live virus. F The mutations destroying the unfavourable restriction sites were used as cloning markers.

Fig. 2

The replication and transcription of the SARS-CoV-2 replicon. The subgenomic RNAs from 293T cells transfected with replicon plasmid were examined using reverse-transcription PCR. The extension time of 1 min (A) and 5 s (B) were performed and the PCR products were detected with DNA agarose electrophoresis. The lengths of PCR fragments which could be amplified theoretically by different primers were depicted in the Table (C). Note that the fragments undetected by PCR were highlighted with red color. D The total RNAs from A549, BHK-21, DLD-1, HEK293T, Huh-7, SY-SH5H, and Vero E6 transfected with the replicon plasmid were reversely transcribed and the cDNA samples were subjected to qRT-PCR. The relative expressions of subgenomic RNAs of each cell line were presented. E Comparisons of the subgenomic RNA composition among Vero E6 expressing replicon, SARS-CoV-2-infected Vero E6 cells, and respiratory tract samples from ten SARS-CoV-2 infected patients. The relative proportion of each subgenomic RNA species was calculated by counting signature leader-body junction sequence for each subgenomic RNA species based on total RNA sequencing of the various samples, respectively.

Fig. 3

The mutations of viral non-structural proteins decrease the synthesis of subgenomic RNAs of the replicon. A The replicon with mutant sites in RdRP (S759A/D760A/D761A, SDD), exonuclease (D90A/E92A, DE), and cap N7 methyltransferase (D331A) were confirmed by Sanger sequencing. B HEK293T cells were transfected with wild-type or mutant replicons with RdRP (S759A/D760A/D761A, SDD), exonuclease (D90A/E92A, DE), or cap N7 methyltransferase (D331A). The relative quantity of subgenomic RNAs were analyzed by qRT-PCR. Note that mutations of RdRP (S759A/D760A/D761A, SDD), exonuclease (D90A/E92A, DE), or cap N7 methyltransferase (D331A) in replicon drastically decreased the synthesis of subgenomic RNAs.

Fig. 4

SARS-CoV-2 replicon functions as an efficient drug screening system. A The reporter gene, mNeonGreen or luciferase, were inserted between SacII and AscI to obtain the constructs of pBAC-nCoV-Replicon-mNeonGreen (Rep-NG) and pBAC-nCoV-Replicon-Luciferase (Rep-Luci). The selection gene, puromycin, was inserted in AscI obtain the constructs of pBAC-nCoV-Replicon-puromycin (Rep-Puro). B HEK293T cells were transfected with wild-type or indicated mutant pBAC-nCoV-Replicon-Luci (Rep-Luci) and pRL-TK. 48 h post-transfection, the cells were subjected to the Dual-Luciferase^® Reporter (DLR™) Assay. HEK293T cells transfected with Rep-NG (C) and Rep-Luci (E) were left untreated or treated with Remdesivir (RDV) at 1 µmol/L. The cells transfected with Rep-NG were fixed and observed under microscope (C) and the green cells were counted in four independent areas (D). The cells transfected with Rep-Luci were subjected to Dual-Luciferase^® Reporter (DLR™) Assay (E). HEK293T cells transfected with Rep-Luci were treated with various drugs, Remdesivir (F), Ritonavir (G), Lopinavir (H) and Carmofur (I) with known inhibitory effect on the replication of SARS-CoV-2 at 8–9 different concentrations. The values of IC50 of each inhibitor were analyzed with Graphpad Prism.