A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation

The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral evaluation. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3000 times higher luminescence than MOCK control cells at 24 h post-electroporation, indicating robust translation and RNA replication of the replicon. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 μM, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 μM). Taken together, this system could be applied to the safe and effective antiviral evaluation without using infectious SARS-CoV-2. Because this is a PCR-based and transient replicon system, further improvement including the establishment of stable cell line must be achieved.

shows the detailed information of the fragments. The viral RNA extracted from the culture fluid of SARS-CoV-2-infected Vero E6 cell was used as a template for RT-PCR. Table S1 shows the primer sets used for the amplification of above-described eight fragments (Fig. 1D). The fragments were assembled in a two-step ligation: (1) all the eight fragments were digested with BsaI, followed by the ligation of two adjacent fragments (e.g. F1 and F2 for F1-2) to produce four assembled fragments; (2) the ligated fragments were gel extracted and mixed, followed by a further ligation to construct the full-length replicon DNA. The size of the successfully ligated replicon DNA was 23.2 kb (Fig. 1E). In vitro transcription using the replicon DNA produced multiple bands (Fig. 1F). Of these bands, the highest band might represent the full-size replicon (indicated by arrow). Because the biggest size of RNA marker was only 8 kb, the estimation of the size of RNA transcripts was not accurate.
Characterization of a SARS-CoV-2 replicon. The in vitro transcribed RNA was directly electroporated (without gel purification) into CHO-K1, BHK-21, or HEK-293T cells to determine the most robust replicon system. In the CHO-K1 cell, the HiBiT signals started to increase as early as 4-6 h post-transfection (hpt), indicating translation and replication of the replicon ( Fig. 2A). At 24-48 hpt, the signals increased by more than 3000 times than the MOCK control. However, the signals decreased at 72 hpt, indicating degradation of the N-HiBiT protein. The BHK-21 and HEK-293T cells showed less HiBiT signals over time ( Figure S1). Thus, the CHO-K1 cell was the most suitable cell line for the robust replication of the replicon, and used for the subsequent experiments.
Subsequently, the kinetics of the replicon RNA in the transfected cells were examined using quantitative reverse transcription PCR (qRT-PCR). The mRNA encoding N gene was also electroporated as a non-replicative RNA control 9 . A decrease was observed for both RNAs at 4 hpt, indicating RNA degradation (Fig. 2B). At 6-24 hpt, the quantity of replicon RNA started to increase, whereas that of the non-replicative RNA showed a (B) Kinetics of the RNA copy. The CHO-K1 cells were electroporated with 10 μg of either the replicon RNA or the non-replicative N gene mRNA. RNA copy numbers were subsequently measured using qRT-PCR. The results were expressed as relative RNA copy number compared to that at 2 hpt. Multiple t-tests were performed for determining the statistical significance. A p-value < 0.05 was considered to be statistically significant. NS not significant. (C) The detection of N protein by IFA. The CHO-K1 cell was electroporated with 5 μg of replicon RNA. The cells were fixed with 4% paraformaldehyde, followed by permeabilization with 0.5% Triton-X. The expression of N protein was detected using anti-N mAb and goat-anti-mouse IgG conjugated with Alexa Fluor 488. Nucleus was stained by DAPI. (D) The detection of NSP8 protein by IFA. The expression of NSP8 protein was detected using anti-NSP8 mAb and goat-anti-mouse IgG conjugated with Alexa Fluor 488. . The NSP8 protein localized not only to the reticular pattern but also to dots ( Figure S3) 14,15 . The ratio of viral protein positive cell was less than 1%. These data indicated that the replicon was successfully constructed and replicative. Antiviral evaluation. Next, we tested if this RNA replicon could be used for antiviral evaluation. Remdesivir, an RdRp inhibitor effective for SARS-CoV-2, was used as a control compound. In total, 10 μM of remdesivir significantly inhibited the translation and replication of the replicon, whereas dimethyl sulfoxide (DMSO) control did not (Fig. 3A). The 50% inhibitory concentration (IC 50 ) and 50% cytotoxicity concentration (CC 50 ) values were calculated to 0.29 μM and more than 50 μM, respectively (selectivity index [SI] > 172.4) (Fig. 3B). The IC 50 value estimated using our replicon system was about 2.6 times lower than the previously reported IC 50 (0.77 μM) 16 . A previous study infected Vero E6 with infectious SARS-CoV-2 in the presence of remdesivir, and quantified the virus released in the supernatant by qRT-PCR at 48 h post-infection 16 . The differences of our replicon assay and previous infectious SARS-CoV-2 assay including cell line (CHO or Vero), incubation time (24 h or 48 h), and action point of analysis (only translation and RNA replication or whole viral replication steps) might cause the difference in IC 50 . Indeed, the difference of the cell line caused different IC 50 values of remdesivir 17 . Nevertheless, the result was generally consistent with the previous report, thus demonstrating that our replicon system could be used for antiviral evaluation.

Discussion
SARS-CoV-2 is an emergent threat worldwide. A high throughput and safe antiviral evaluation system is urgently needed to identify the anti-SARS-CoV-2 compound, which has not yet been developed. Several plasmid-based SARS-CoV-2 replicons have been reported 18,19 . Here, we reported a SARS-CoV-2 replicon system with PCR amplicon-based strategy. The advantage of this system is its technical simplicity. Additionally, this system enabled us to produce a replicon without generating genetically modified E. coli. Thus, bacteriotoxic elements in the SARS-CoV-2 genome do not affect the construction of the replicon. However, the PCR-based strategy might be inferior to the plasmid-based strategy in terms of the yield of replicon RNA and usability of genome modification. Additionally, PCR-based replicon might contain the undesired mutations, which are undetectable by Sanger sequence. Nevertheless, this PCR-based replicon system offered an alternative way over plasmid-based replicon, especially in the resource-limited settings.
The cells electroporated with the replicon RNA showed more than 3,000 times higher luminescence as compared to the MOCK control cells at 24 hpt ( Fig. 2A). However, the replicon RNA copy was increased by only 1.5 times at 24 hpt compared to that at 2 hpt (Fig. 2B); this could be attributed to RNA degradation and low positive rate of successful replication of the replicon in the cells (< 1%). Nevertheless, the replicon showed a significant increase in the RNA copy compared to the non-replicative control RNA at 24 hpt, indicating RNA replication. The replicon RNA copy as well as the HiBiT signal started decreasing at 48 hpt and 72 hpt, respectively, indicating low stability of the replicon. This discrepancy was considered due to the higher stability of N-HiBiT protein www.nature.com/scientificreports/ than that of the replicon RNA. The mRNA levels of IFN-β and Mx1 were increased in replicon-transfected cells ( Figure S2), suggesting the role of interferon signaling on the rapid decrease of replicon RNA. Further studies such as testing the replicon transfection in CHO-K1 cells upon inhibition of IFN-β production and/or Jak-STAT signaling are needed to validate this notion. In this study, CHO-K1, BHK-21, and 293T cells were used because these cell lines were used for the construction of coronavirus replicon and coronavirus protein expression 3,4 . However, only CHO-K1 supported the robust replication of the replicon. Interestingly, even BHK-21, which is defective of IFN production, showed less replication of the replicon 20 . This was not attributed to the electroporation efficacy because the difference of input RNA in each cell was observed to exhibit an enhancement within twofold ( Figure S4A). CHO-K1 showed the highest fold enhancement of the replicon copy number at 24 hpt among the three cell lines ( Figure S4B). The obtained data suggested that the host factors other than IFN might be related to the lower replication of SARS-CoV-2 replicon in BHK-21 and 293T cells, although further analysis is needed.
We chose to fuse HiBiT-tag to the N protein because subgenomic mRNA-encoding N was the most abundantly produced mRNA during the replication of coronavirus 21 . This study demonstrated that the insertion of HiBiT-tag at the C-terminus of N protein did not disrupt the RNA replication. This finding could be applied to the construction of HiBiT-tagged reporter infectious virus 22 . We had also tried to fuse HiBiT-tag at the N-terminus of N protein ( Figure S5A, S5B, and Table S2). The luminescence of the replicon with N-terminal HiBiT was 10 times lower than that with C-terminal HiBiT at 24 hpt ( Figure S5C). The N protein is involved in not only nucleocapsid formation, but also RNA replication such as helicase activity and genome-length negativestrand RNA synthesis 23,24 . Although the N-terminus of N protein was not associated with either RNA binding or dimerization 25 , the modification of the N-terminus might affect the replication efficacy. Alternatively, the position of HiBiT-tag in the N protein might have affected the sensitivity of HiBiT assay.
This replicon system can be used not only for antiviral evaluation but also for the analysis of SARS-CoV-2 ORF1ab function in terms of RNA replication. SARS-CoV-1 replicon was applied to the functional analysis of non-structural proteins encoded in ORF1 4 . Nowadays, several mutations have been observed in the replication complex regions because of worldwide pandemic 26 . For example, the virological meaning of ORF1ab 4715L mutation positively correlated to a high fatality rate remains unknown 27 . This system would help to shed light on the enigmatic SARS-CoV-2 RNA replication mechanism.
The disadvantages of this system were that our replicon was a transient expression system, which was not a high throughput system. The cell line stably carrying the replicon gene needs to be established by inserting the antibiotic resistance gene such as puromycin N-acetyl-transferase into the replicon genome 3 . Additionally, our replicon lacks the structural genes including S, E, and M. Thus, this system cannot be used for the compounds acting on receptor binding, virus entry, encapsidation, and virus release. These targets could be covered by using a single-round infectious pseudo-type reporter virus usable in the BSL-2 laboratory 28 .
In conclusion, we reported a SARS-CoV-2 replicon that can be applied to antiviral evaluation without using infectious virion. Further improvement of this replicon system would accelerate the antiviral screening and help to identify the novel drug candidates for COVID-19.
The construction of a SARS-CoV-2 replicon DNA. The viral RNA extracted from the culture fluid of SARS-CoV-2-infected Vero E6 cell (provided by the National Institute of Infectious Diseases, Japan) was reverse transcribed into cDNA by the SuperScript III First Strand Synthesis system (Thermo Fisher Scientific) with random hexamer primers. The fragments were amplified by primer sets (Table S1) and high-fidelity PCR with the Platinum SuperFi II DNA polymerase (Thermo Fisher Scientific). F8 was generated by the overlap PCR of F8A and F8B fragments to insert the HiBiT-tag at the C-terminus of N gene (Table S1). The overhang sequences after BsaI digestion were designed based on the ligase fidelity viewer program (available at the New England Biolabs website).
RNA transcription, electroporation, and luminescence quantification. The  www.nature.com/scientificreports/ ally, a SARS-CoV-2N gene mRNA was in vitro transcribed following a report 9 . After removing the DNA template following the manufacturer's protocol, RNA was extracted by phenol-chloroform and isopropanol precipitated. The pelleted RNA was washed once with 70% ethanol, dried by air, and dissolved in 40 μl of DEPC-treated water. The RNA was electrophoresed using DynaMarker RNA High for Easy Electrophoresis (BioDynamics Laboratory. Inc.) for the rough quality check.
The RNA was electroporated using NEPA21 electroporator (Nepagene). The cells were trypsinized and washed twice with Opti-MEM (Thermo Fisher Scientific). The washed cells (1 × 10 6 cells) were mixed with 5 μg of replicon RNA in 100 μL of Opti-MEM. Electric pulses were given by NEPA21. The parameters for BHK-21 and CHO-K1 cells were as follows: voltage = 145 V; pulse length = 5 ms; pulse interval = 50 ms; number of pulses = 1; decay rate = 10%; polarity + as poring pulse and voltage = 20 V; pulse length = 50 ms; pulse interval = 50 ms; number of pulses = 5; decay rate = 40%; and polarity + / − as transfer pulse. The parameters for 293 T cell was same as above except voltage 150 V and pulse length of 2.5 ms for poring pulse. After electroporation, the cells were seeded as 1.5 × 10 4 cells/well in a 96-well plate. At various time points post-transfection, the cells were lysed with 25 μl of Nano-Glo HiBiT lytic detection system (Promega) plus 25 μl of PBS. The luminescence signal was detected by CentroPRO LB962 (Berthold Technologies).