Versatile SARS-CoV-2 Reverse-Genetics Systems for the Study of Antiviral Resistance and Replication

Abstract

The COVID-19 pandemic continues to threaten healthcare systems worldwide due to the limited access to vaccines, suboptimal treatment options, and the continuous emergence of new and more transmissible SARS-CoV-2 variants. Reverse-genetics studies of viral genes and mutations have proven highly valuable in advancing basic virus research, leading to the development of therapeutics. We developed a functional and highly versatile full-length SARS-CoV-2 infectious system by cloning the sequence of a COVID-19 associated virus isolate (DK-AHH1) into a bacterial artificial chromosome (BAC). Viruses recovered after RNA-transfection of in vitro transcripts into Vero E6 cells showed growth kinetics and remdesivir susceptibility similar to the DK-AHH1 virus isolate. Insertion of reporter genes, green fluorescent protein, and nanoluciferase into the ORF7 genomic region led to high levels of reporter activity, which facilitated high throughput treatment experiments. We found that putative coronavirus remdesivir resistance-associated substitutions F480L and V570L-and naturally found polymorphisms A97V, P323L, and N491S, all in nsp12-did not decrease SARS-CoV-2 susceptibility to remdesivir. A nanoluciferase reporter clone with deletion of spike (S), envelope (E), and membrane (M) proteins exhibited high levels of transient replication, was inhibited by remdesivir, and therefore could function as an efficient non-infectious subgenomic replicon system. The developed SARS-CoV-2 reverse-genetics systems, including recombinants to modify infectious viruses and non-infectious subgenomic replicons with autonomous genomic RNA replication, will permit high-throughput cell culture studies-providing fundamental understanding of basic biology of this coronavirus. We have proven the utility of the systems in rapidly introducing mutations in nsp12 and studying their effect on the efficacy of remdesivir, which is used worldwide for the treatment of COVID-19. Our system provides a platform to effectively test the antiviral activity of drugs and the phenotype of SARS-CoV-2 mutants.

Keywords: GFP; RNA virus; SARS-CoV-2; molecular clone; nanoluciferase; polymerase; remdesivir; replicon.

Figure 1

Reverse-genetics system for SARS-CoV-2. (a) Diagram of the bacterial artificial chromosome (BAC), in which the complete genome (from four cloned fragments as indicated) of SARS-CoV-2 was inserted. Individual open reading frames (ORFs) are depicted as arrow boxes. 5′ and 3′: untranslated regions; RZ: HDV-ribozyme; NotI: restriction site. (b) In vitro transcription of the full-length SARS-CoV-2 plasmid, 40 ng of RNA transcript loaded per well. 1: ssRNA ladder; 2: DNase-treated RNA transcripts; 3: RNA transcripts; 4: 1kb DNA ladder. The white arrow indicates full-length RNA transcripts. (c) to the left of the dotted line, representative peak infectivity titers (TCID₅₀/mL) of the second passage virus. To the right, RNA titers (as GE/mL) of the same samples measured by E-sequence specific RT-qPCR. (d) Representative pictures of infected immunostained Vero E6 cells. (e) Virus propagation kinetics after infection at MOI 0.01. Graphs indicate the number of infected cells determined by immunostaining (y-axis) at different time points (x-axis). Data are shown as mean and standard error of the mean. Patient (blue): virus isolated from a COVID-19 patient [22]; clone (red): virus rescued from RNA transcribed from the clone illustrated in (a); blank (black): culture medium.

Figure 2

Clone reporter systems for SARS-CoV-2. (a,b) Representative images of SARS-CoV-2 Spike, SARS-CoV-2 Nucleocapsid, and GFP channels, together with an overlay of all channels including the nucleus, from a time-course infection experiment (MOI 0.01) of (a) wild-type clone (WT) and (b) GFP-reporter viruses, taken with a 40× objective. (c) Titration of nLuc and Fluc reporter activity of SARS-CoV-2 reporter viruses with initial infectivity titers of 5.5 and 5.3 Log TCID₅₀/mL, respectively. The values of relative light units (RLU) measured at 24 h were subtracted from negative control wells and normalized to the values obtained from the 10⁻¹ virus dilution. The data points represent mean of quadruplicates; error bars represent standard error of the mean. For some data points the differences are too small to plot error bars.

Figure 3

Susceptibility to remdesivir. (a) Antiviral activity of remdesivir against the patient and the clone viruses (passage 2 viruses). (b) Antiviral potency of remdesivir against the nLuc reporter virus measured 24, 48, and 72 h after treatment using a luciferase assay. The graphs show the non-linear regression curve of the number of SARS-CoV-2 infected cells treated with different remdesivir concentrations normalized to non-treated controls. The dots and circles represent the mean of triplicates; error bars represent standard error of the mean. For some data points the differences are too small to plot error bars. EC₅₀ values (µM) inferred from the regression are shown in parenthesis.

Figure 4

Analysis of SARS-CoV-2 nsp12 polymerase mutants. (a) Peak infectivity titers (TCID₅₀/mL) of second passage SARS-CoV-2 viruses with specified nsp12 substitutions and of the parental wild-type clone (WT). (b) SARS-CoV-2 RNA titers of the same samples measured by E-sequence specific RT-qPCR. (c,d) Concentration–response curves of infections from the constructs depicted in (a) and (b) treated with different remdesivir concentrations. The dots represent the mean, and standard errors of the mean are shown as lines. EC₅₀ values (µM) inferred from the regression analysis are shown in parenthesis for each construct. For some data points the differences are too small to plot error bars.

Figure 5

Generation of SARS-CoV-2 subgenomic replicon systems. (a) Structure of the replicon constructs, with the nLuc ORF7a insertion highlighted in green. (b) Luciferase activity of the different replicons after transfection with 2 μg of RNA. (c) Luciferase activity after transfection with different amounts of RNA transcripts. (d,e) Luciferase activity of the different SARS-CoV-2 nLuc systems under treatment with remdesivir (RDV) at various concentrations. Remdesivir was added 32 h prior to transfections and re-applied 1-h post transfection. Mock, transfection without any RNAs. The datapoints represent the mean of three independent transfections. Each transfection was performed in triplicate, and the mean of each transfection was used to calculate the values shown here; error bars represent standard errors of the mean. For some data points the differences are too small to plot error bars.