An optimized high-throughput SARS-CoV-2 dual reporter trans-complementation system for antiviral screening in vitro and in vivo

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still epidemic around the world. The manipulation of SARS-CoV-2 is restricted to biosafety level 3 laboratories (BSL-3). In this study, we developed a SARS-CoV-2 ΔN-GFP-HiBiT replicon delivery particles (RDPs) encoding a dual reporter gene, GFP-HiBiT, capable of producing both GFP signal and luciferase activities. Through optimal selection of the reporter gene, GFP-HiBiT demonstrated superior stability and convenience for antiviral evaluation. Additionally, we established a RDP infection mouse model by delivering the N gene into K18-hACE2 KI mouse through lentivirus. This mouse model supports RDP replication and can be utilized for in vivo antiviral evaluations. In summary, the RDP system serves as a valuable tool for efficient antiviral screening and studying the gene function of SARS-CoV-2. Importantly, this system can be manipulated in BSL-2 laboratories, decreasing the threshold of experimental requirements.

Keywords: Antiviral evaluation; BSL-2; Mouse model; RDP; SARS-CoV-2.

Fig. 1

Design and construction of SARS-CoV-2 ΔN reporter RDP. A Schematic of SARS-CoV-2 genome, the open reading frames (ORFs), structural proteins (S, E, M, N) and accessory proteins (3a, 6, 7, 8, 10) are indicated. B Schematic of SARS-CoV-2 ΔN-reporter RDP genome and fragments for yeast assembly (bottom panel). The reporter gene was placed downstream of the regulatory of N to replace the N sequence. C Electrophoresis of the seven DNA fragments. Seven purified DNA fragments (about 500 ​ng) were run on a 1% agarose gel. The DL15000 DNA marker is indicated. D Multiplex PCR screening and identification of yeast clones carrying SARS-CoV-2 ΔN-reporter RDP genome. Six sets of primers were used to ensure the presence of all junctions between the adjacent fragments. The expected junction PCR product sizes are indicated in the right panel. The 1 ​kb DNA ladder is indicated. E Gel analysis of full-length (FL) and N RNA transcripts. About 500 ​ng in vitro transcribed RNAs were analyzed on a 0.8% native agarose gel. The DL15000 marker is indicated. Arrow indicates the genome full-length RNA transcripts. Circles denotes the truncated RNA transcripts. F Construction of a lentivirus transfer plasmid encoding mCherry and N protein, and RT-PCR analyses were performed on Caco-2-N cells and Caco-2 ​cells to detect the N gene. G The mCherry expression in serially passaged Caco-2-N cells for 20 passages. The percentage of P1, P5, P10, P15 and P20 mCherry positive cells are presented. The results are presented as means ​± ​SD from three replicates. About 10⁴ ​cells were counted for each replicate. Statistical significance was determined using one-way analysis of variance (ANOVA). ∗∗∗∗P ​< 0.0001.

Fig. 2

RDP rescue, and optimization and stability of SARS-CoV-2 reporter RDP. A The GFP signal of electroporated Caco-2-N cells. Scale bar is indicated. B Continuously passaging the SARS-CoV-2 ΔN-GFP, SARS-CoV-2 ΔN-Nluc-GFP and SARS-CoV-2 ΔN-GFP-HiBiT RDPs on Caco-2-N cells for five rounds, and the titer of P0–P5 RDPs were determined. C RT-PCR analysis of the stability of the reporter genes of P0, P1 P3 and P5 SARS-CoV-2 ΔN-GFP, SARS-CoV-2 ΔN-Nluc-GFP and SARS-CoV-2 ΔN-GFP-HiBiT RDPs, respectively (top panel). The SARS-CoV-2 M gene was set as an internal reference (bottom panel). The scheme of the RT-PCR was indicated at the left, and the agarose gels of PCR products were shown at the right. GFP, Nluc-GFP (NG), GFP-HiBiT (GH) and SARS-CoV-2 M (M) fragments were performed as controls, respectively. The 1 ​kb DNA ladder is indicated.

Fig. 3

Stability and characterization of SARS-CoV-2 ΔN-GFP-HiBiT RDP. A The fluorescence of the SARS-CoV-2 ΔN-GFP-HiBiT wild type (WT) and mutant (D614G and S-BA.5.2) RDPs infected cells. Scale bar, 200 ​μm. B–D Kinetics of titer and (E) the area under the curve were analyzed. Statistical significance was determined using one-way analysis of variance (ANOVA). ∗∗∗∗P < 0.0001; ns, not significant, P ​> ​0.05. Kinetics of the luminescence (F), SARS-CoV-2 E mRNA level (G) and sub-genome E (sgE) mRNA copy (H). Caco-2 ​cells were infected with authentic SARS-CoV-2 WT (authentic WT). Caco-2-N cells were infected with the SARS-CoV-2 ΔN-GFP-HiBiT wild type (WT) and mutant (D614G and S-BA.5.2) RDPs, respectively. Caco-2-N cells were infected at MOI 0.05. At the given time points, cells were harvested for luciferase signal measurement. Viral sgE mRNAs in the infected cells and viral/RDP yield in the infected cell supernatants was quantified by RT-qPCR.

Fig. 4

High-throughput antiviral evaluation using SARS-CoV-2 and SARS-CoV-2 ΔN-GFP-HiBiT RDP. A Antiviral drugs evaluation assay scheme in a 96-well format. Remdesivir (left panel), nirmatrelvir (middle panel) and REGEN10933 (right panel) were evaluated. B Comparison of drugs evaluation between SARS-CoV-2 and SARS-CoV-2 ΔN-GFP-HiBiT WT RDP by RT-qPCR. The drugs were evaluated in SARS-CoV-2 ΔN-GFP-HiBiT WT and mutant RDPs by measuring luminescence signal (C) and GFP signal (D), respectively. Cytotoxicity of these drugs to Caco-2-N cells was measured by CCK-8 assays. The left and right Y-axis of the graphs represent mean % inhibition of luciferase signal and cytotoxicity of the drugs, respectively. The experiments were done in triplicates. Dates are normalized to infected cells without antiviral drugs or antibody. The four-parameter dose-response curve was fitted using nonlinear regression method. The IC₅₀ was calculated by Prism GraphPad and indicated.

Fig. 5

Construction of a preliminary SARS-CoV-2 ΔN-GFP-HiBiT RDP infection model through lentivirus-N. A The experiment schemes. The K18-hACE2 KI mice were intranasally infected with 2 ​× ​10⁹ copies lentivirus-N and feed for another 14 days. Then the lentivirus-N infected mice were intranasally infected with 4 ​× ​10⁶ TCID₅₀ SARS-CoV-2 ΔN-GFP-HiBiT RDPs. Mouse body weight was monitored for up to 7 days post-infection and all the mice were sacrificed at 7 days post-infection. B Body weight change of lentivirus-N infected mice and lentivirus-N ​+ ​SARS-CoV-2 ΔN-GFP-HiBiT RDP infected mice. C WB was used to detect the expression of N protein in lung of lentivirus-N infected mice. D The N mRNA expression levels in lentivirus-N infected mice tissues were measured using RT-qPCR. E–F The sgE mRNA expression levels and viral genome copies in RDP-infected mice tissues were measured using RT-qPCR. G The viral titers in lungs of RDP-infected and uninfected lentivirus-N-transduced mice were measured using TCID₅₀ method. Each dot represents one mouse (n ​= ​5). The relative RNA expression was normalized to GAPDH. H–I Immunofluorescence analysis and pathological changes in lungs of mock, lentivirus-N and lentivirus-N ​+ ​SARS-CoV-2 ΔN-GFP-HiBiT RDP infected mice, respectively. J Heat map of cytokines and chemokines in lungs of infected mice.

Fig. 6

Evaluate the antiviral drug and neutralizing antibody in vivo. A The scheme of evaluation of remdesivir (RDV) and REGN10933 (REGN) in vivo. The time of RDV and REGN treatment was indicated in the illustration. The RDP and authentic SARS-CoV-2 infected mice are indicated at top and bottom panel, respectively. B The viral RNA copies in the lung were measured at 7 dpi. Statistical significance was determined using two-tailed, unpaired t-test. ∗∗P ​< ​0.01; ns, not significant, P ​> ​0.05. C Section of paraffin embedded lungs from mock, RDV and REGN group mice were stained with hematoxylin/eosin. The scale bar is indicated.

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