In order to rapidly construct SARS-CoV-2-GFP replicon plasmids and bypass the instability of viral gene fragments in E. coli, transformation-associated recombination (TAR) cloning in S. cerevisiae was chosen as a strategy, as this approach has been successfully used for the rapid construction of MERS-CoV and SARS-CoV-2 infectious clones (Nikiforuk et al. 2016; Thi et al. 2020). The overall reverse genetic strategy is illustrated in Fig. 1. This replicon includes untranslated 5′ and 3′ ends of the viral genome, the replicase gene ORF1ab with an NSP1 deletion, since this suppresses host gene expression and may be toxic in transfected cells (Shi et al. 2020; Yuan et al. 2020), as well as the nucleocapsid N gene that has been reported to be required for viral RNA synthesis, a partial sequence of ORF8 and transcription regulatory sequence (TRS) of the N gene and ORF10. In order to enable easy quantification of viral replication and selection of replicon-containing cells, a GFP-blasticidin deaminase fusion gene (GFP-BlaR) was inserted to substitute for the spike gene under the control of the TRS of the spike gene. This strategy has been successfully used by others to construct SARS-CoV-derived replicon cell lines (Ge et al. 2007). This whole replicon genome was placed under control of a cytomegalovirus (CMV) promoter. Hepatitis delta virus ribozyme (HDVRz), bovine growth hormone termination, and polyadenylation (BGH) sequences were also added to the 3′ region of Poly A sequences to ensure the production of integral viral RNAs.
This whole reverse genetic system was based on the virus strain EPI_ISL_403929 (GISAID database, which is identical to GenBank accession number MN908947). The designed replicon genome was divided into 26 fragments (F1–F26, Fig. 1A, also see Supplementary Materials). Most of the fragments were generated by RT-PCR from viral RNA, and the rest including F1, F23, F24, and F26 were generated by PCR from chemically synthesized cDNAs or other vectors (Fig. 1A). Each fragment contains a ~30 bp overlap with its neighboring fragments and was then stepwise assembled by overlap extension PCR. Finally, nine sub-genomic fragments were obtained and transformed into S. cerevisiae MaV203 cells with linear pYES1L vector for homologous recombination (Fig. 1B). pYES1L vector is a YAC-BAC shuttle plasmid that can be used as a cloning vector for the simultaneous and seamless assembly of DNA fragments in S. cerevisiae cells. Furthermore, the BAC-based design overcomes the stability issues when transformed into E. coli cells. This plasmid also has been reported successfully used in constructing MERS-CoV infectious clone previously (Nikiforuk et al. 2016). After growth for three days at 30 ℃, recombinant yeast colonies began to appear on CSM-Trp agar plates (Fig. 2A). Four yeast colonies were randomly picked and screened for three replicon fragments (F12–F13; F18–F19; F23–F25) by yeast colony PCR, and the results showed that all four colonies were positive for the three tested fragments (Fig. 2B), indicating the high efficiency of the TAR cloning system. Afterward, the assembled constructs in positive yeast colonies were transferred to E. coli, and five colonies were then picked and screened for replicon fragments (F16–F17) by PCR. Again, the results indicated that all of the five colonies were positive (Fig. 2C). Then, these plasmids were isolated from E. coli. The finally obtained plasmid was screened for all nine fragments used in yeast homologous recombination by PCR, and the results showed that all of them were present in the resulting plasmid and were of anticipated sizes (Fig. 2D). Next, the assembled SARS-CoV-2-GFP replicon plasmid was sequenced, and three point-mutations were identified in the whole sequence (Table 1), two with amino acid mutations in the coding region of NSP2 and NSP3, and one with no amino acid change in the coding region of NSP12. These individual mutations occurred during RT-PCR and overlap extension PCR since the large size of the SARS-CoV-2 genome.
Figure 2. Verification of a SARS-CoV-2-GFP replicon plasmid. A Recombinant yeast colonies on CSM-Trp agar plates. B Transformed yeast clone was tested for fragments F12–F13, F18–F19, and F23–F25 by yeast colony PCR. C E. coli clones transformed with yeast plasmid were tested for F16–F17 by PCR. D Recombinant SARS-CoV-2-GFP replicon plasmid was tested for all nine sub-genomic fragments used in yeast homologous recombination by PCR.
Encoded protein Genomic position Nucleotide change Amino acid change NSP2 397 C → T Thr → Met NSP3 6969 T → C Leu → Pro NSP12 14,559 G → A None
Table 1. Sequence analysis of SARS-CoV-2-GFP replicon.
To verify whether our SARS-CoV-2-GFP replicon plasmid was valid, this plasmid was transfected into 293 T cells. Thirty-six hours post-transfection (hpt), tGFP expression was monitored using fluorescence microscopy, and obvious green fluorescence was visually observed in SARS-CoV-2-GFP-transfected cells but not in non-transfected cells (Fig. 3A). Then, Western blot was used to further confirm the expression of the tGFP-BlaR gene and viral nucleocapsid (N) protein, and as shown in Fig. 3B, both the tGFP-BlaR fusion gene and N protein could be detected in SARS-CoV-2-GFP-transfected cells. The tGFP-BlaR fusion gene was expressed as a 40 kDa protein as expected. Collectively, these results demonstrated that the SARS-CoV-2-GFP replicon could replicate and be transcribed in transfected cells. To optimize the time point for following drug evaluation, kinetics study of tGFP-BlaR expression after SARS-CoV-2-GFP replicon transfection was carried out and the results showed that the tGFP-BlaR signal began to be detected at 12 hpt and peaked at 36 hpt (Fig. 3C). Therefore, thirty-six hours post-transfection was chosen as the time point to evaluate the activity of antiviral drugs in the following study.
Figure 3. SARS-CoV-2-GFP replicon transfection assay. A Fluorescence microscopy monitoring tGFP expression. 293 T cells were transfected or nontransfected (BLANK) with SARS-CoV-2-GFP replicon, and 36 h post-transfection, cells were fixed, observed and photographed, the nuclei were stained with Hoechst dye, scale bar: 100 µm. B 293 T cells were transfected or nontransfected (BLANK) with SARS-CoV-2-GFP replicon, and 36 h post-transfection, cells were lysed followed by Western blot to monitor the expression of tGFP-BlaR, Nucleocapsid protein (N), and Actin was used as a loading control. C Kinetics of tGFP-BlaR expression, 293 T cells were transfected with SARS-CoV-2-GFP replicon and were lysed at indicated hours post-transfection (hpt) and tGFP-BlaR expression was monitored by Western blot, Actin was used as a loading control.
To explore the utility of the SARS-CoV-2-GFP replicon for antiviral drug evaluation, we tested the dose-responsive effect of two previously reported coronavirus replication inhibitors. The first inhibitor was E64-D, which is a cysteine proteinase inhibitor and blocks replicase polyprotein processing in viral replication (Kim et al. 1995). This was reported to work well in both SARS-CoV tissue culture systems and replicon systems (Ge et al. 2007; Wang et al. 2008; Yount et al. 2003). E64-D treatment at a concentration of 0.1 mg/mL and 0.2 mg/mL significantly reduced the tGFP fluorescence in SARS-CoV-2-GFP replicon-transfected 293 T cells at 36 h post-transfection when observed under a fluorescence microscope (Fig. 4A). This reduction in tGFP expression was also evident by Western blot for tGFP-BlaR expression (Fig. 4B). The second inhibitor tested was remdesivir, which is an adenosine analogue and can incorporate into nascent viral RNA chains to result in pre-mature termination (Pruijssers et al. 2020). Remdesivir was recently recognized as a promising antiviral drug against a wide array of RNA viruses including the Ebola virus, SARS-CoV, and MERS-CoV (Sheahan et al. 2017), as well as SARS-CoV-2 in cell culture (Wang et al. 2020) and animal models (de Wit et al. 2020), and is currently under clinical development for the treatment of Ebola virus infection and SARS-CoV-2 infection (Hashemian et al. 2020). Consistent with other reports, remdesivir inhibited tGFP expression in SARS-CoV-2-GFP replicon-transfected 293 T cells and exhibited an obvious dose dependence by both fluorescence microscopy observation (Fig. 4C) and Western blot for tGFP-BlaR expression (Fig. 4D). Collectively, our results demonstrated the suitability of this SARS-oV-2-GFP replicon system for evaluating and studying candidate antiviral agents.
Figure 4. Inhibitory effect of E64-D and remdesivir on a SARS-CoV-2-GFP replicon. A, B 293 T cells were transfected with SARS-CoV-2-GFP replicon, and ten hours post-transfection, cells were treated or nontreated (BLANK) with E64-D at indicated concentrations. Thirty-six hours post-transfection, cells were observed and photographed by fluorescence microscopy, the nuclei were stained with Hoechst, scale bar: 100 µm (A) or lysed for Western blot monitored the expression of tGFP-BlaR, actin was used as a loading control (B). C, D 293 T cells were transfected SARS-CoV-2-GFP replicon, and cells were treated or nontreated (BLANK) with remdesivir at indicated concentrations just before transfection. Thirty-six hours post-transfection, cells were observed and photographed using fluorescence microscopy, the nuclei were stained with Hoechst, scale bar: 100 µm (C), or lysed for Western blot for tGFP-BlaR, Actin was used as a loading control (D).
A good drug evaluation system usually reflects a well dose-responsive relationship, and the half-maximal effective concentration (EC50) is often used to evaluate the antiviral effect of a specific compound. Although E64-D and remdesivir exhibit dose-dependent antiviral effect on the replicon system, we explored this replicon system to estimate the EC50 value of the two compounds. We used two methods to quantification tGFP-BlaR expression and calculated EC50 respectively to test the reproducibility of the system. First, we determined GFP fluorescence intensity by a microplate reader and drawing dose-responsive curve with the fluorescence intensity value (Fig. 5A and 5B), then the EC50 value of the two compounds was calculated as 36.3 μmol/L and 8.93 μmol/L respectively (Fig. 5A and 5B). We also evaluated cell viability using CCK8 assay in parallel in 293 T cells and demonstrated that although cytotoxic effects were observed at the highest concentration of E64-D and remdesivir in the assay, no cytotoxic effects were observed at the EC50 concentration of both two drugs (Fig. 5A and 5B). Next, we determined tGFP-BlaR expression by Western blot assay, as shown in Fig. 5C and 5E, the expression of tGFP-BlaR decreased gradually when the dose of E64-D and remdesivir increased. By drawing dose–response curves using the ratio of tGFP-BlaR/β-actin gray values from Western blot, the EC50 value of E64-D and remdesivir was determined as 47.60 μmol/L and 6.76 μmol/L (Fig. 5D and 5F). These results are very similar to the previous results determined by fluorescence intensity value and the slight difference is due to different detection methods. Taken together, these results further demonstrated that a SARS-CoV-2-GFP replicon could be reliably used to evaluate antiviral drugs.
Figure 5. Determining the EC50 of E64-D and remdesivir using a SARS-CoV-2-GFP replicon. A, B 293 T cells were transfected with SARS-CoV-2-GFP replicon, E64-D and remdesivir were added to culture media 10 h post transfection or just before transfection respectively, the green fluorescence intensity was determined thirty-six hours post-transfection, and dose responsive curves were drawn and EC50 value of E64-D (A) and remdesivir (B) was calculated by four-parameter nonlinear regression respectively. Cell viability was determined by CCK8 assay in parallel on 293 T cells. The experiments were done in triplicates. C–F Replicon transfection and drug treatment were performed as mentioned in (A, B), thirty-six hours post-transfection, Western blot for tGFP-BlaR expression was carried out with E64-D (C) and remdesivir treated cells (E). EC50 value of E64-D (F) and remdesivir (G) as determined by dose response curves using the ratio of tGFP-BlaR/β-actin gray values with different concentration and calculated by four-parameter nonlinear regression. The experiments were done at least two times.
In order to expand the application scope of our replicon system, we constructed a dual-reporter SARS-CoV-2-GFP-Luc replicon using our original SARS-CoV-2-GFP replicon. We engineered a firefly luciferase (FLuc) in frame with ORF8 of the viral genome (Fig. 6A). The previously mentioned transformation-associated recombination cloning protocol was carried out, but the fragment F23–F24 used in SARS-CoV-2-GFP recombination was substituted with F23 and F24-FLuc. Ten sub-genomic fragments together with linearized pYES1L were transformed into S. cerevisiae MaV203 cells to obtain a reassembled replicon plasmid (SARS-CoV-2-GFP-FLuc). The resulting plasmid was also screened for all fragments used in recombination by PCR, and our results demonstrated that all were present (Fig. 6B). Then, the SARS-CoV-2-GFP-FLuc replicon was transfected into 293 T cells, and luciferase activity was detected thirty-six hours post-transfection. The results showed a ~ 6000-fold increase in luciferase activity in SARS-CoV-2-GFP-FLuc replicon-transfected cells (Fig. 6C), demonstrating this replicon worked well. Similarly, kinetics study monitoring luciferase activity after SARS-CoV-2-GFP-Fluc replicon transfection was carried out, and the result showed 36 hpt was also the peak time point of luciferase activity (Fig. 6D). Next, this SARS-CoV-2-GFP-FLuc replicon was transfected into 293 T cells, and the EC50 value of E64-D was determined. Thirty-six hours post-transfection, cells were lysed, and dose response curves were drawn with the luciferase activity values or tGFP-BlaR/β-actin gray values determined by Western blot respectively. The EC50 of E64-D was derived to be 48.95 μmol/L by luciferase signals (Fig. 6E), and was 36.67 μmol/L determined by tGFP-BlaR signals (Fig. 6F). The results determined by the two indicators are comparable to each other, indicating the utility of this SARS-CoV-2-GFP-FLuc replicon for drug evaluation as well.
Figure 6. Construction a SARS-CoV-2-GFP-FLuc replicon and determination of the EC50 of E64-D. A Diagram of SARS-CoV-2-GFP-FLuc replicon. B Verification of a SARS-CoV-2-GFP-FLuc replicon plasmid. Recombinant SARS-CoV-2-GFP-FLuc replicon plasmid was tested for all sub-genomic fragments used in yeast homologous recombination by PCR. C Luciferase activity in nontransfected (BLANK) or SARS-CoV-2-GFP-FLuc replicon-transfected or 293 T cells, the luciferase activity was determined 36 h post transfection. D Kinetics study of luciferase activity after SARS-CoV-2-GFP-FLuc replicon transfection, 293 T cells were transfected with SARS-CoV-2-GFP-Fluc replicon and luciferase activity was determined at indicated hours post-transfection (hpt). E EC50 value of E-64D was determined using a dose response curve drawn based on luciferase activity in SARS-CoV-2-GFP-FLuc-transfected 293 T cells using four-parameter nonlinear regression. F EC50 value of E-64D was determined using a dose response curve drawn based on the ratio of tGFP-BlaR/β-actin gray values with different concentration and calculated by four-parameter nonlinear regression, the experiments were done at least two times.