Rapid generation of HCoV-229E and HCoV-OC43 reporter viruses and replicons for antiviral research

Abstract

Introduction: The large size of coronavirus genome, along with the instability of certain genomic sequences, makes the construction of reverse genetics for coronaviruses particularly challenging. The rapid development and application of reverse genetics systems for coronaviruses require further exploration.

Methods: Using transformation-associated recombination (TAR) cloning in yeast and the in vitro CRISPR-Cas9 system, reverse genetics systems of two mild coronaviruses HCoV-OC43 and HCoV-229E were rapidly established. Antiviral assays, high-content imaging, and NanoLuc luciferase assays were used to characterize reporter viruses and replicon systems.

Results: We rapidly assembled infectious clones for two mild coronaviruses, HCoV-OC43 and HCoV-229E, using transformation-associated recombination (TAR) cloning in yeast. The infected clones could stably express the mGreenLantern reporter gene. We further generated T7 promoter-driven RNA replicon of HCoV-229E and CMV promoter-driven DNA replicon of HCoV-OC43, with the readout of NanoLuc luciferase activity. The effectiveness of these tools for antiviral study was evaluated using the broad-spectrum RNA-dependent RNA polymerase inhibitor remdesivir, exhibiting high sensitivity, efficiency, and convenience.

Discussion: The application of yeast-based TAR cloning significantly facilitates the rapid assembly of large viral genome, and the establishment of HCoV-OC43 and HCoV-229E reverse genetics systems provides valuable platforms for studying the biology and developing antivirals against coronaviruses.

Keywords: TAR cloning; antiviral; human coronavirus; replicon; reporter virus; reverse genetics system.

Figure 1

Schematic representation of the construction of full-length infectious clones of 229E-mGreen and OC43-mGreen reporter viruses. (A) Schematic of wild-type 229E and chimeric 229E-Green genomes. The open reading frames (ORFs) of the nonstructural, structural, and accessory genes are indicated. The mGreenLantern gene (mGreen) was inserted to replace the ns4a gene. (B) Schematic of wild-type OC43 and chimeric OC43-mGreen genomes. The ORFs of nonstructural, structural, and accessory genes are indicated. The mGreen gene was inserted upstream of the HE gene to replace ns2a. The N-terminal 18 amino acids of the ns2a were maintained. Both 229E and OC43 genomes are divided into nine fragments (F1-F9), with approximately 130 bp of overlap between adjacent fragments.

Figure 2

Recovery and characterization of 229E-mGreen and OC43-mGreen reporter viruses. (A) Workflow of TAR cloning and reporter virus rescue. The DNA fragments with overlapping junctions were assembled through homologous recombination in yeast, and full-length cDNA clones was extracted from yeast and electroporated into E. coli competent cells. Virus rescue was initiated by transfecting BHK-21 cells with purified plasmids, followed by amplification in susceptible cells. (B) Cytopathic effects (CPE) of 229E-mGreen and OC43-mGreen reporter viruses in Huh7 at 24 h and HRT-18 cells at 72 h, respectively. The parental wild-type viruses were used as a control. Scale bar, 400 μm (top) or 80 μm (bottom). (C) Huh7 cells were infected with 229E-WT and 229E-mGreen for immunofluorescence staining of nucleocapsid (N) protein (MOI 0.02, 24 h). (D) HRT-18 cells were infected with OC43-WT and OC43-mGreen for immunofluorescence staining of nucleocapsid (N) protein (MOI 0.01, 72 h). The images were captured using a fluorescence microscope. Scale bar, 400 μm.

Figure 3

Growth property of recovered reporter viruses. (A) Growth kinetics of 229E reporter virus. Huh7 cells were infected with 229E-WT and 229E-mGreen (MOI 0.01). Supernatants were collected at the indicated time points, and viral titers were determined in huh7 cells by focus forming assay. (B) Focus-forming morphology of 229E reporter virus. Huh7 cells were infected with 229E-WT and 229E-mGreen (MOI 0.02, 24 h). The mGreen-positive fluorescence (top) and immunostaining of nucleocapsid (bottom) images were captured and the areas (square micrometer) of foci were quantified using a CTL ImmunoSpot analyzer. The mean sizes of >100 foci per virus with positive standard deviations was shown. (C) Growth kinetics of OC43 reporter virus. HRT-18 cells were infected with OC43-WT and OC43-mGreen (MOI 0.005). Supernatants were collected at the indicated time points, and viral titers were determined in HRT-18 cells by focus forming assay. (D) Focus-forming morphology of OC43 reporter virus. HRT-18 cells were infected with OC43-WT and OC43-mGreen (MOI 0.01, 72 h). The mGreen fluorescence (top) and immunostaining of nucleocapsid (bottom) images were captured and the areas (square micrometer) of foci were quantified using a CTL ImmunoSpot analyzer. The mean sizes of >100 foci per virus with positive standard deviations was shown. * : P < 0.05; ** : P < 0.01; *** : P < 0.001, ns : not statistically significant.

Figure 4

Stability analysis of the mGreen reporter gene during serial passaging. (A, B) Growth kinetics of the indicated passages (P1, P5, P10) of reporter viruses. Huh7 cells were infected with 229E-mGreen (P1, P5, P10) at a MOI 0.01 (A). HRT-18 cells were infected with OC43-mGreen (P1, P5, P10) at a MOI 0.005 (B). Supernatants were collected at the indicated time points and viral titers were determined in respective cells by focus-forming assay. (C, D) P1, P5, and P10 stocks of 229E-mGreen (MOI 0.02, 24 h) (C) and OC43-mGreen (MOI 0.01, 72 h) (D) were used to infect Huh7 and HRT-18 cells, respectively. The foci formed upon infection with the indicated passages (P1, P5, P10) of reporter viruses were analyzed for the expression of mGreen (top) and nucleocapsid protein (bottom). Images were captured using a CTL ImmunoSpot analyzer, and the percentage of mGreen-positive foci in nucleocapsid-positive foci were calculated. (E, F) Analysis of the genetic stability of the inserted mGreen gene after 10 passages in susceptible cells. Viral RNA was extracted from culture supernatants of P1, P5 and P10, and RT-PCR was performed using primer pairs flanking the reporter gene region. The amplified products were resolved by 1% agarose gel electrophoresis.

Figure 5

Validation of an image-based antiviral assay using reporter viruses. (A) The procedure of antiviral assay with mGreen reporter viruses. Cells in 96-well plates were pre-treated with three-fold dilutions of remdesivir for 60 minutes, followed by infection in the presence of the compound. The infection duration was optimized for detecting fluorescent protein expression (229E-mGreen, MOI 0.02, 36 h; OC43-mGreen, MOI 0.005, 96 h). After fixation, cells were stained with DAPI, and images were captured using the Operetta CLS High-Content Analysis System. Fluorescent images from each well were analyzed to calculate the percentage of infected cells. (B) Representative images of Huh7 or HRT-18 cells infected with 229E-mGreen or OC43-mGreen, respectively. Cells were pre-treated with 2 μM remdesivir or DMSO prior to infection. (C, D) Antiviral effect of remdesivir on 229E-mGreen (C) or OC43-mGreen (D). The cells pre-treated with remdesivir were infected with reporter viruses, and mGreen-positive cells were quantified (229E-mGreen 36 h; OC43-mGreen, 96 h). Cell viability following treatment with remdesivir was determined. The IC₅₀ and CC₅₀ values were calculated.

Figure 6

Construction and characterization of 229E and OC43 replicon systems. (A) Schematic diagram showing the construction of 229E and OC43 replicon systems. The full-length infectious clones (non-structural, structural, and accessory genes as shown in Figure 1 ) were digested using an in vitro CRISPR-Cas9 system to delete the ORFs of structural (S, E, and M) and accessory genes, which were subsequently replaced with the NanoLuc luciferase (NLuc) gene. The replicons were driven by T7 promoter (T7p), and the hepatitis delta virus ribozyme (HDVr) and T7 terminator (T7 ter) were added after the poly (A) tail. For OC43, cytomegalovirus (CMV) promoter-driven DNA replicon was also constructed. (B) HeLa cells were electroporated with replicon RNAs, and NLuc activity was determined at the indicated timepoints. (C) HeLa cells were electroporated with replicon RNAs in the presence of 20 μM remdesivir (RDV) or 0.1% DMSO control, and NLuc activity was measured at 36 h post-transfection. (D) HEK-293T cells were transfected with CMV-driven OC43 replicon plasmid, and NLuc activity was measured at indicated timepoints. Mock transfection and OC43 replicon harboring NSP12 mutations (D760N/D761N) were used as controls. (E) HEK-293T cells were pre-treated with 20 μM RDV or 0.1% DMSO control for 1 h, followed by transfection with WT OC43 replicon or its NSP12 mutant for 6 (h) The medium was then replaced with fresh medium containing 20 μM RDV or 0.1% DMSO for another 18 h for measurement of NLuc activity. *** : P < 0.001, **** : P < 0.0001, ns : not statistically significant.