A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses

Abstract

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.

Fig. 1. Generation of SARS-CoV-2 by CPER and characterization of properties of recovered viruses in cells and mice.

a Schematics of SARS-CoV-2 genome and overlapping SARS-CoV-2 fragments amplified from SARS-CoV-2 cDNA and circularized with a linker fragment containing the last 20 nucleotides of SARS-CoV-2 3′UTR, 30As, hepatitis delta virus ribozyme (HDVr), SV40 pA signal for transcription termination, spacer sequence, CMV promoter, and first 37 nucleotides of SARS-CoV-2 5′UTR. The resultant SARS-CoV-2 CPER product was then directly transfected into HEK293T cells, then cocultured with Vero E6 cells for virus recovery. b Agarose gel electrophoresis of PCR-amplified SARS-CoV-2 fragments 1–6 and the linker fragment showing a representative image of three experimental repeats. c Representative plaque morphologies of the wild-type (WT) SARS-CoV-2_QLD02 isolate and CPER-recovered viruses (CPER1 and CPER2) stained using crystal violet at 2 dpi (top) and Immuno-plaque assay (iPA) with anti-spike protein monoclonal antibody at 14 hpi (bottom). d Growth kinetics of WT (red) and CPER-generated viruses (green and light blue) over a 3-day time course in Vero E6 cells infected at a multiplicity of infection MOI = 0.01, n = 2 independent experiments with three replicates in each, statistical analysis was performed by two-way analysis of variance with Tukey’s multiple comparisons test against WT virus. Mean values for each virus at each time point are shown ± SD. e End-point virus titers of nasal turbinates and lung tissues from JAX K18-hACE2-transgenic mice infected intranasally with 8 × 10⁴ FFU/mouse of WT SARS-CoV-2_QLD02 isolate (red) and CPER-recovered viruses (blue). At 5 days post infection, mice were sacrificed, and virus titers were determined by TCID₅₀ assay on Vero E6 cells. For statistical analysis between WT and CPER viruses, unpaired t-test with Welch’s correction was used, p values are two-sided. Mean values for each treatment are shown ± SD. For WT virus, three biological replicates were used, for CPER-generated virus six biological replicates were used, results are from one experiment. f Full lungs from mice infected with WT virus, representative CPER virus, or infected (mock), harvested at 5 days post infection and stained with hematoxylin and eosin (H&E). Scale bar is 5 mm. g Selected H&E stained images of lung sections from mice infected with WT or representative CPER virus showing sloughing of the bronchial epithelium as indicated by the arrowhead, bronchi occluded with edema (O) and red blood cells (R). h Additional features of SARS-CoV-2 infection in lungs of infected mice showing the collapse of alveolar spaces (A) and edema. The scale bar is 100 µm. Representative images from f–h are from three independently analyzed samples for each treatment. Source data for d and e are provided in the Source Data file.

Fig. 2. Generation by CPER and characterization of SARS-CoV-2 D614G mutant virus and ZsGreen reporter virus.

a Schematics of D614G mutant virus genome and overlapping fragments used to introduce D614G mutation by CPER. Fragment 5 is split into subfragments 5A and 5B with their overlapping region incorporating D614G mutation. b Agarose gel electrophoresis of PCR-amplified SARS-CoV-2 fragments representative of at least three experiments. c Representative plaque morphologies of wild-type (WT) SARS-CoV-2_QLD02 isolate, CPER-recovered D614G mutant virus, and SARS-CoV-2_QLD935 isolate naturally harboring the D614G mutation. Infected cells were stained with crystal violet at 3 dpi. d Sanger sequencing of CPER-generated D614G mutant virus (top) and wild-type SARS-CoV-2_QLD02 cDNA (bottom). e Growth kinetics of SARS-CoV-2_QLD02 isolate (red circle), CPER-generated D614G mutant virus (black triangle), and SARS-CoV-2_QLD935 (orange square) isolate over a 3-day time course in Vero E6 cells infected at MOI = 0.01, n = 2 independent experiments with three replicates in each. Statistical tests to examine differences in growth kinetics between virus isolates were analyzed using a two-way ANOVA. The differences between each time point were analyzed with Tukey’s multiple comparisons test. Adjustments to p values were not made for multiple comparisons. ****p ≤ 0.0001. Bar graph is shown as mean values ± SD. f Schematics of SARS-CoV-2_{ΔORF7a-ZsGreen} reporter virus and overlapping fragments used to generate this virus. Fragment 6 is split into two subfragments, 6A and 6B to generate a 95 codon deletion in ORF7a (deleted codons 14-108), and ZsGreen gene is inserted in the place of this deletion. Images of ZsGreen fluorescence of Vero E6 cells infected with MOI = 0.1 of CPER-generated reporter virus taken at 40× magnification (g) and 100× magnification (h). Representative images from g and h are from three independently analyzed samples from each treatment and two independent experiments. i Representative plaque morphologies of wild-type (WT) SARS-CoV-2_QLD02 isolate and CPER-generated ZsGreen reporter virus stained with crystal violet at 2 dpi. j Growth kinetics of SARS-CoV-2_QLD02 (WT, blue) and CPER-generated reporter virus (ZsGreen, green) over a 3-day time course in Vero E6 cells infected at MOI = 0.01. n = 2 independent experiments with three replicates in each. Graph is shown as mean values ± SD. k Sanger sequencing showing positions 23603-23620 of the SARS-CoV-2 QLD02 isolate corresponding to the polybasic furin cleavage site (RRAR) in the spike protein. ZsGreen1, amplified in Vero E6 cells, shows ambiguous peak at 23616 (indicated with the arrowhead) corresponding to variable deep sequencing data at this position. ZsGreen2, amplified and passaged on Vero E6-TMPRSS2 cells, shows no change to the furin cleavage site over three passages. l RT-PCR with the CoV-6F/6R primer pair of fragment 6 containing ZsGreen insertion compared to the parental QLD02 isolate showing retention of insertion over three passages in Vero E6-TMPRSS2 cells. The ZsGreen2 virus amplifies a 4094 nt fragment, whereas the WT QLD02 virus amplifies a 3689 nt fragment. Gel electrophoresis is representative of at least three experiments. m Median fluorescence intensity (MFI) values from flow cytometry analysis (Supplementary Fig. 4) normalized to viral titers (MFI/log₁₀(FFU/mL)) of ZsGreen2 virus over three passages in Vero E6-TMPRSS2 cells. Statistical analysis was an Unpaired t-test with Welch’s correction, p values are two-tailed and shown above comparison. Data shown as individual biological replicates from three independent passage experiments (n = 3) ± SD. Source data for e, j, and m are provided in the Source Data file. The scale bar for g and h is 100 and 10 µm, respectively.

Fig. 3. Generation by CPER and characterization of wild-type and mutant Ross River viruses, and their application for studying virus–receptor interactions.

a Schematics of RRV T48 strain genome, overlapping fragments, and linker fragment used for CPER assembly. OpIE2—insect promoter, CMV—mammalian promoter. b Agarose gel electrophoresis of PCR-amplified RRV fragments and linker fragment. A representative of at least three experiments is shown. c Representative plaque morphologies of wild-type (WT) RRV viruses in Vero cells (top left) or A. albopictus C6/36 cells (top right) as well as of CPER-recovered RRV viruses generated using CMV promoter (bottom left) or OpIE2 promoter (bottom right). Infected cells were stained with crystal violet at 3 dpi. d Growth kinetics of WT (black square) and CPER-generated (white circle) RRV viruses in Vero 76 cells over a 3-day time course infected at MOI = 0.1, n = 3 independent experiments with two replicates in each, statistical analysis to compare each time point for each of the mutants against CPER WT virus was performed by two-way analysis of variance with Tukey’s multiple comparisons test. Adjustments were not made for multiple comparisons. Mean values of the three independent experiments ± SD of the mean are shown. e Schematics of overlapping RRV fragments amplified from RRV_T48 cDNA and introduced furin site mutations at the E3/E2 cleavage junction. Modified furin cleavage sites are introduced from Influenza virus H5N1 (RRV_H5) and Semliki forest virus (RRV_SFV) through the overlapping regions in fragments 4 and 5. Location of furin cleavage is indicated by an arrowhead. f Representative foci and plaque morphologies of CPER-generated RRV furin site mutants as shown from immuno-plaque assay at 12 hpi (top) and crystal violet staining at 3 dpi (bottom). g Growth kinetics of CPER-generated RRV furin mutants (RRV_SFV; white square, RRV_H5; black square, RRV_CPER; red circle), in mammalian (Vero) cells (left) and A. albopictus C6/36 cells (right), n = 3 independent experiments with two replicates in each. Statistical analysis was performed by two-way analysis of variance with Tukey’s multiple comparisons test. Adjustments were not made for multiple comparisons. ****p ≤ 0.0001. Mean values of the three independent experiments ± SD of the mean are shown. h Binding assay for E1/E2 RRV mutants using purified virus and recombinant hMXRA8 receptor and represented as equilibrium-binding affinity (Kd) at 37 °C. Data used to generate the nonlinear curve are available from Source Data file, data represent two independent experimental replicates (n = 2). Summary table presented here as bar graph. i Position of the RRV mutants shown in h on the reported CHIKV-hMXRA8 structure (PDB:6J08). The heterotrimers of E1 and E2 are shown in white and blue surface representation, respectively. hMXRA8 is shown in green surface representation in the left panel, and the binding footprint shown in orange on the right. Mutated residues are indicated in pink, with labels indicating the amino acid for CHIKV at each position and corresponding residue for RRV in brackets. Source data for d, g, and h are provided in the Source Data file.

Fig. 4. Generation of mouse and human noroviruses and insect-specific mesonivirus CASV by CPER and their characterization.

a Schematics of human norovirus (HuNoV) and murine norovirus (MNV) genomes and of overlapping fragments and linker fragment used for CPER assembly. b Agarose gel electrophoresis of PCR-amplified MNV, HuNoV, and linker fragments showing a representative image of two experiments for HuNoV and at least three for MNV. c Virus titers quantitation of MNV CPER-transfected NIH3T3 cells at 3 days post transfection (MNV_CPER P₀) and of virus further amplified in RAW264.7 cells at 3 days post infection (MNV_CPER P₁), results indicate four independent transfections and subsequent infections. d Representative plaque morphologies of wild-type MNV (MNV_WT) and CPER-recovered MNV (MNV_CPER). e Immunoblot analysis of mock, MNV_WT and MNV_CPER infected NIH3T3 cell lysates probed with anti-MNV-VP1 (top) and anti-actin (bottom) antibodies blot are representative of two experiments. f HuNoV genomic RNA (gRNA) quantitation by RT-qPCR analysis of supernatants from CPER-transfected NIH3T3 cells at 3 and 72 h post transfection from two independent experiments (n = 2). g Schematics of Casuarina virus (CASV) genome and overlapping fragments as well as linker fragment used for CPER assembly. h Agarose gel electrophoresis of PCR-amplified CASV fragments and linker fragments showing a representative image of more than three independent experiments. i Immunofluorescence analysis by confocal microscopy of A. albopictus C6/36 cells transfected with CPER products generated by three different CPER cycling conditions (CPER1, CPER2, CPER3), showing representative images of one independent experiment. CPER3 has been reproduced in two separate independent experiments. The scale bar is 50 µm. Viral antigen is visualized by staining with anti-CASV ORF2a protein monoclonal antibody C.9D7 and cell nuclei stained with Hoechst 33342. j Growth kinetics of CPER-generated (blue square) and WT CASV (red circle) in C6/36 cells infected at MOI = 0.1, n = 2 independent experiments were conducted with three replicates. Statistical analysis was performed by two-way analysis of variance with Tukey’s multiple comparisons test. Graphs in c, f, and j are mean values ± SD. Source data for c, f, and j are provided in the Source Data file.