key: cord-0935316-84pif64i
authors: Zhang, Hu; Fischer, Douglas K.; Shuda, Masahiro; Moore, Patrick S.; Gao, Shou‐Jiang; Ambrose, Zandrea; Guo, Haitao
title: Construction and characterization of two SARS‐CoV‐2 minigenome replicon systems
date: 2022-02-19
journal: J Med Virol
DOI: 10.1002/jmv.27650
sha: b18a929de9a0f3d6576c914b86c596f525bb4f15
doc_id: 935316
cord_uid: 84pif64i

The ongoing COVID‐19 pandemic severely impacts global public health and economies. To facilitate research on severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virology and antiviral discovery, a noninfectious viral replicon system operating under biosafety level 2 containment is warranted. We report herein the construction and characterization of two SARS‐CoV‐2 minigenome replicon systems. First, we constructed the IVT‐CoV2‐Rep complementary DNA template to generate a replicon messenger RNA (mRNA) with nanoluciferase (NLuc) reporter via in vitro transcription (IVT). The replicon mRNA transfection assay demonstrated a rapid and transient replication of IVT‐CoV2‐Rep in a variety of cell lines, which could be completely abolished by known SARS‐CoV‐2 replication inhibitors. Our data also suggest that the transient phenotype of IVT‐CoV2‐Rep is not due to host innate antiviral responses. In addition, we have developed a DNA‐launched replicon BAC‐CoV2‐Rep, which supports the in‐cell transcription of a replicon mRNA as initial replication template. The BAC‐CoV2‐Rep transient transfection system exhibited a much stronger and longer replicon signal compared to the IVT‐CoV2‐Rep version. We also found that a portion of the NLuc reporter signal was derived from the spliced BAC‐CoV2‐Rep mRNA and was resistant to antiviral treatment, especially during the early phase after transfection. In summary, the established SARS‐CoV‐2 transient replicon systems are suitable for basic and antiviral research, and hold promise for stable replicon cell line development with further optimization.

previously characterized coronaviruses ( Figure 1 ). 6, 7 Approximately two-thirds of the viral genome encodes an open reading frame (ORF), ORF1ab, which is the only ORF directly translated into proteins from the full-length viral genome. ORF1ab is translated into two polypeptides, specifically PP1a and PP1ab, via a programmed −1 ribosome frameshift mechanism. 8 The polypeptides undergo autoproteolysis to release a number of nonstructural proteins (NSPs) including proteases, RNA-dependent RNA polymerase (RdRp, also known as NSP12), as well as other NSPs required for viral RNA replication and transcription. 9 The translated replicase and transcriptase proteins engage the viral genome to assemble the replicasetranscriptase complex (RTC) on endoplasmic reticulum membrane, forming a membranous compartment. 10 Within the membranous compartment, the RTC completes viral replication and transcription.

The latter involves a transcription regulatory sequences (TRS)mediated discontinuous RNA synthesis, which results in that all the viral transcripts contain the same 5ʹ-leader and 3ʹ-terminal sequences from the full-length genome. 11, 12 Transcription of the 3ʹterminal genome by viral RTC generates a nested set of subgenomic messenger RNAs (mRNAs) that encode structural proteins and accessory genes. Viral structural proteins include the spike (S), membrane (M), envelop (E), and nucleocapsid (N) proteins required for virion assembly. 13 Establishing SARS-CoV-2 infection and/or replication systems in the laboratory setting is indispensable for basic and antiviral research. Reverse genetics represents a powerful approach for this purpose, by which recombinant viruses are engineered to express reporter genes to facilitate antiviral drug screening, and attenuated viruses or viral replicons can be generated to provide a safer biocontainment environment for studying potentially lethal infectious viruses. 14, 15 Self-amplifying viral replicon RNA systems that recapitulate the core events of intracellular viral genome synthesis, but avoid the production of infectious viral particles, have been successfully utilized in virus research and antiviral drug discovery, as exemplified by the hepatitis C virus (HCV) replicon. 16, 17 Previous studies have used various approaches to assemble coronavirus replicons. Some made use of in vitro ligation to assembly the replicon complementary DNA (cDNA), followed by in vitro transcription (IVT) to generate replicon mRNA [18] [19] [20] [21] ; others used transformation associated recombination (TAR) or a bacterial artificial chromosome (BAC) to assemble replicon cDNA and then produce replicon mRNA by IVT. 22, 23 The IVT mRNA were then transfected into target host cells to initiate replicon RNA replication. While these IVT RNAbased approaches have been demonstrated to work, they also face the technical challenge of producing full-length viral RNA of ∼25 kb, especially when scale-up production is required. For example, instead of full-length RNA, shorter RNA species have been shown to be the major transcripts produced by IVT of replicon cDNA. 20, 24 A recent study reported a DNA-launched BAC system that appeared to be an effective and simple approach to generate SARS-CoV-2 replicon RNA with high signal; however, this system was refractory to antiviral drug treatment. 25 Furthermore, for unknown reasons, all the reported SARS-CoV-2 replicon RNA systems were transient in cells, and a stable replicon cell line has not been established so far.

Here, we report our work on SARS-CoV-2 minigenome replicon construction and optimization. First, we used the prokaryotic T7 promoter-driven IVT to generate IVT-CoV2-Rep mRNA with a nanoluciferase (NLuc) reporter from the replicon cDNA, followed by cell transfection. The experimental results demonstrated that IVT-CoV2-Rep worked in a variety of host cell lines.

Although the replicon signal was low and transient for several days, it could be completely abolished by known SARS-CoV-2 protease and RdRp inhibitors. Furthermore, our data suggested that the transient phenotype of IVT-CoV2-Rep was unlikely due to host innate antiviral responses. In addition, we have also developed a BAC plasmid-based replicon (BAC-CoV2-Rep), which supports the transcription of full-length replicon mRNA under the control of a eukaryotic cytomegalovirus-immediate early (CMV-IE) promoter upon transfection into host cells. The BAC-CoV2-Rep system exhibited a much stronger and longer replicon signal compared to the IVT-CoV2-Rep version. We also found that a portion of the BAC-CoV2-Rep NLuc reporter signal was derived from spliced replicon mRNA and resistant to antiviral treatment, especially during the early phase after transfection. Taken together, the BAC-CoV2-Rep system holds promise for basic and antiviral research, as well as stable replicon cell line development with further optimization.

The African green monkey kidney epithelial cell line Vero, Chinese hamster ovary cell line CHO-K1, and baby hamster kidney cell line BHK-21, were purchased from the American Type Culture Collection.

The human hepatoma cell line Huh7.5 was kindly provided by Dr.

Charles Rice (Rockefeller University). 26 The human lung epithelial cell line A549DKO with protein kinase R (PKR) and ribonuclease L (RNase L) double knockout was kindly provided by Dr. Bernard Moss (National Institute of Allergy and Infectious Diseases/National Institutes of Health [NIAID/NIH]). 27 Cells were maintained in Dulbecco's modified Eagle's medium:nutrient mixture F-12 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

As illustrated in Figure 1 , the 5ʹ-portion of SARS-CoV-2 genome (Isolate USA_WA1/2020) was split into multiple cDNA fragments.

The cDNA fragment-containing plasmids pUC57-F1, pCC1BAC-F2, pCC1BAC-F3, and pUC57-F4 were kindly provided by Dr. Pei-Yong Shi (University of Texas Medical Branch). 24 Table S1 . The 5ʹ ends of F5ʹ forward and reverse primers were designed to contain the Type IIS restriction enzyme Esp3I site. The TRS element before S ORF (TRS-S) in SARS-CoV2 genome was included in the reverse primer (F5ʹr). The F5ʹ PCR fragment was cloned into pSC-A-amp kan (Agilent) vector to obtain plasmid pSC-A-F5ʹ. Plasmid pUAS-NanoLuc expressing NLuc was a gift from Dr. Robert Campbell (Addgene plasmid # 87696). 28 The LbN DNA fragment containing the NLuc and blasticidin S-resistance gene (BSR) fusion gene ORF (Lb) and SARS-CoV-2 N ORF in tandem was partially synthesized by Genscript and assembled with a NLuc-containing PCR fragment amplified from pUAS-NanoLuc. An 85-nt sequence containing TRS-N upstream of N ORF was included in LbN. A poly(A) tail of 33 nt was placed at the 3ʹ terminus of LbN. The Esp3I site was introduced to the 5ʹ and 3ʹ termini of LbN as well. LbN fragment was subcloned into pcDNA3.1/V5-His (V81020; Invitrogen) to obtain pcDNA3.1-LbN. Plasmid pcDNA6B-N expressing the C-terminally FLAG-tagged SARS-CoV-2 N protein was a gift from Dr. Pei-Hui Wang (Shandong University) and Dr. Yuan Liu (Cornell University). 29 All fragments were sequenced and confirmed to be correct according to the SARS-CoV-2 full-length genome reference sequence (NCBI GenBank accession number: NC_045512). 

The IVT-CoV2-Rep mRNA was in vitro transcribed by the mMES-SAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific) according to the manufacturer's instruction with minor modifications.

One microgram of IVT-CoV2-Rep cDNA was subjected to IVT. Cap analog to GTP ratio was set to 1:1. The reaction was incubated overnight at 30°C. After removal of DNA template by RQ1 RNase-Free DNase treatment, RNA was precipitated by LiCl. Pelleted RNA was washed once with 70% ethanol, dried by air and dissolved in 30 μl diethylpyrocarbonate-treated water. The purified RNA samples were quantified by Nanodrop spectrophotometer (Thermo Fisher Scientific) and subjected to native and denature agarose gel electrophoresis for quality assessment.

The EcoRV-linearized pcDNA3.1-LbN and XbaI-linearized pcDNA6B-N were used as IVT templates for LbN and N mRNA transcription, respectively, as above described except that the IVT reaction time was reduced to 1 h.

To prepare the riboprobe targeting the F1 fragment region of fulllength SARS-CoV-2 replicon mRNA, a pair of PCR primers, specifically probeF1f and SP6probeF1r (Table S1) , were used to amplify the DNA template from pUC57-F1. The purified PCR product served as the template for SP6 IVT (P1420; Promega) of the radiolabeled F1 riboprobe in the following reaction mixture: 2 μl of purified F1 riboprobe DNA template (500 ng/μl), 4 μl of transcription optimized 5X buffer, 2 μl of 100 mM DTT, 1 μl of RNase inhibitor (20 units/μl), 4 μl of rATP, rGTP, rCTP, and UTP mix (2.5 mM each except for 0.125 mM of UTP), 1 μl of SP6 RNA polymerase (20 units/μl), and 6 μl of [ 32 P]-UTP (10 mCi/ml; PerkinElmer). After incubation of the reaction at 37°C for 1 h, 1 μl of RQ1 RNase-free DNase (1 U/μl) was added to remove the DNA template, and the riboprobe was purified and the radioactivity was measured by a scintillation counter (Per-kinElmer) according to our published protocol. 31 To make the riboprobe targeting the N gene sequence in all the full-length and subgenomic mRNAs of SARS-CoV-2, a 123-bp PCR product amplified by the N gene qPCR primers (Table S1) 

For reverse transcription-quantitative PCR (RT-qPCR) of IVT-CoV2-Rep RNA, the cDNA templates were prepared with 10 ng input RNA, F1-specific RT primer (Table S1, Table S1 .

RT-PCR of total RNA extracted from BAC-CoV2-Reptransfected cells was conducted using splicing-specific RT and PCR primers (Table S1 ). The RT-PCR products were resolved in an agarose gel and the visualized DNA bands were purified and cloned into the pSC-A-amp/kan vector (Agilent) for Sanger sequencing. 

The transfected cells were fixed with 4% paraformaldehyde, followed by permeabilization with 0.5% Triton-X. After blocking with normal immunofluorescence assay buffer (10% fetal bovine serum + 2% bovine serum albumin in phosphate-buffered saline solution), the cells were first incubated with primary antibodies against SARS-CoV-2 N protein (40143-MM05; Sinobiological), followed by incubation with fluorescence-labeled secondary antibodies. The cells were mounted in a mounting medium containing 4ʹ,6-diamidino-2-phenylindole.

Fluorescence images were acquired by a fluorescence microscope (EVOS M5000; Thermo Fisher Scientific).

SARS-CoV-2 antiviral compounds remdesivir, GC376, and EIDD-1931 were purchased from SelleckChem. Remdesivir and GC376

were dissolved in dimethyl sulfoxide (DMSO) to make 10 mM stocks. EIDD-1931 was dissolved in water to make a 10 mM stock. RIG-I ligand 5ʹ triphosphate hairpin RNA (3p-hpRNA) was purchased from InvivoGen. PKR inhibitor C16 was purchased from Bio-Techne.

Data are provided as the mean ± standard deviation. Calculations and graphs were generated using GraphPad Prism 9.0. Student's t test was used to determine the statistical significance. A p value less than 0.05 (p < 0.05) was considered statistically significant.

The workflow of our design of the SARS-CoV-2 replicons is schematically illustrated in Figure 1 . The designed minigenome replicon contains the viral NSP genes (ORF1a and ORF1b) and the N gene required for coronavirus RNA replication. 32 Then, the IVT-CoV2-Rep cDNA was subjected to T7 promotermediated IVT. The IVT products exhibited a predominant species around 1.5 kb with a long smear in a native agarose gel electrophoresis ( Figure 2D ). We also ran the IVT products in a denaturing gel and conducted Northern blotting using riboprobes targeting the N ORF and the F1 region, respectively ( Figure S2) . A strong hybridization signal at a low molecular weight position was detected by the F1 probe but not the N probe, which was likely the nascent IVT products. However, full-length replicon RNA was not detected by either the F1 or the N riboprobes, indicating that the IVT of full-length replicon mRNA was inefficient likely due to the large size of the IVT-CoV2-Rep cDNA template. To examine whether the full-length replicon RNA was produced by IVT, we developed a more sensitive RT-qPCR assay to detect the F1 and N region, respectively (Table S1 , (Table S2) .

To construct BAC-CoV2-Rep, we took advantage of the unique BsiWI and XhoI restriction sites located in the 5ʹ F1 region and 3ʹ N ORF in the IVT-CoV2-Rep construct, respectively. The acceptor plasmid pCC1BAC-CMV-PreSARS2 harbors the tandem CMV-IE and T7 promoters, a portion of the 5ʹ and 3ʹ terminal sequence of SARS-CoV2 genome, and the hepatitis delta virus ribozyme sequence ( Figure 2E ). Therefore, the BsiWI/XhoI-restricted fragment from IVT-CoV2-Rep cDNA was cloned into the corresponding site in the pCC1BAC-CMV-PreSARS2 vector. The positive BAC-CoV2-Rep clone was verified by BsiWI/XhoI double digestion ( Figure 2F ). We then checked whether the observed transient replicon replication could rebound in a prolonged culture of transfected cells.

We chose CHO-K1 and BHK-21 cells for further study based on the relatively higher replicon-NLuc activity produced in these two cell lines. As shown in Figure 3E ,F, THE NLuc activity peaked early but quickly declined to background level within 4 days posttransfection, and remained at background level over 5 days of additional follow-up monitoring, indicating the presence of a quenching host antiviral mechanism(s) or lack of a viral factor(s) responsible for the observed transient phenotype of IVT-CoV2-Rep. 

It has been reported that the cellular pattern recognition receptor RIG-I/MDA5 can sense SARS-CoV-2 double stranded RNA (dsRNA) structures to induce type I/III IFNs, but the virus is able to antagonize the induction of IFNs and subsequent IFN-mediated Janus kinase-signal transducer and activator of transcription antiviral signaling by a battery of viral nonstructural, structural, and accessory proteins. [37] [38] [39] [40] In light of the fact that IVT-CoV2-Rep does not encode viral structural and accessory proteins, we speculated that the transfected replicon RNA and/or replication intermediates might induce cellular innate immunity which eliminates replicon replication.

However, it is also worth noting that among the above tested host cell lines, Huh7.5 possesses a mutationally inactivated RIG-I for IFN production, 41 Vero cells lack the IFN-β gene, 42 Figure 5C ). Furthermore, we tested IVT-CoV2-Rep mRNA in A549DKO cells, a PKR and RNase L double knockout cell line. 27 Nevertheless, the replicon replication remained transient ( Figure 5D ).

Collectively, these results suggested that the cellular innate immunity was not the major determinant of the unsustainable IVT-CoV2-rep replication.

Another plausible explanation for the transient IVT-CoV2-Rep replication might be the low level of full-length IVT mRNA generated for transfection ( Figure S2 and Table S2 ). We, therefore, set out to make use of the pCC1BAC vector system for transcription of the replicon mRNA in cells (Figures 1 and 2 Figure 6B ). We also performed Northern blot analysis to detect the RNA species produced by BAC-CoV2-Rep.

We found that the LbN mRNA could be detected at a low level, although the full-length replicon RNA and N mRNA were undetectable ( Figure 6C) . Altogether, these data demonstrated that the BAC-CoV2-Rep system is superior to the IVT-CoV2-Rep version with respect to reporter signal strength and duration, as well as viral subgenomic mRNA transcription and gene expression.

The BAC-CoV2-Rep-transfected CHO-K1 cells were treated with the same set of antiviral compounds used in the above IVT-CoV2-Rep experiment. However, the reduction of BAC-CoV2-Rep NLuc activity by antiviral treatment was not as significant as that observed in treatment of IVT-CoV2-Rep. Remdesivir only slightly reduced the NLuc level by ∼10% ( Figure 7A ), which is somewhat consistent with a recent study using a similar BAC replicon system. 25 

Considering that the NLuc signal in BAC-CoV2-Rep transfection might come from other sources independent of replicon replication, we speculated that this may be due to mRNA splicing since the initial replicon mRNA is transcribed from BAC-CoV2-Rep plasmid DNA in the cell nucleus, where RNA splicing occurs to transform pre-mRNAs into the mature mRNAs and to generate multiple functional mRNAs or proteins from a single transcript. 51 To test this hypothesis, we designed a pair of primers targeting the 5ʹ untranslational region (UTR) and Lb ORF on the replicon genome to detect potential spliced RNA species that could directly translate Lb ( Figure 8A ). Next, CHO-K1 cells were transfected with BAC-CoV2-Rep or the negative control pcDNA3.1-LbN, and total RNA were extracted and subjected to RT-PCR. As shown in Figure 8B , RT-PCR with the N qPCR primers (Table S1) The ∼550 bp band was then gel purified and cloned into a plasmid for sequencing. After sequencing more than 20 clones, three major splicing RNA species were identified and their splicing sites are described in Figure 8C . Sp1, Sp2, and Sp3 all possess the sequence of NLuc ORF with a variable short 5ʹ untranslated region. The sequences at the identified splicing sites are consistent with the major splice donor and acceptor consensus motif "GT_AG," and there is only one noncanonical 3ʹ acceptor sequence ( Figure 8D ). 52 Taken together, the data demonstrate that BAC-CoV2-Rep mRNA splicing occurs during transient transfection, giving rise to cryptic NLuc reporter signals independent of replicon replication. Reverse genetics approaches were used to generate SARS-CoV-2 infectious clone and replicon systems immediately after the whole viral genome sequence became publicly available. 24, 40 Due to the large size of SARS-CoV-2 genome, the viral cDNA genome was designed as split fragments of ∼5 kb each with suitable type IIS restriction sites located at the junctions, then the chemically synthesized fragments were subjected to restriction enzyme digestion and ligated into cDNA in vitro. 23, 37 In addition, a prokaryotic promoter sequence, such as T7, and a poly(A) sequence were typically placed at the 5ʹ and 3ʹ termini of the replicon cDNA, respectively. To rapidly acquire the replicon mRNA, most researchers performed IVT of the assembled replicon cDNA. 20, 40 Alternatively, others further cloned the aforementioned cDNA into BAC vectors to obtain a more consistent and scalable production of replicon cDNA template for IVT. 19, 23, 25 It is worth noting that the above reverse genetics approaches have been successfully used previously in generating other coronavirus replicons. 55 A recent study employed the TAR apparatus to assemble large overlapping SARS-CoV-2 cDNA fragments to create a replicon DNA plasmid in the yeast Saccharomyces cerevisiae. 22, 56 Another group reported a SARS-CoV-2 replicon transfection system consisting of four plasmids expressing ORF1ab proteins and the minimally required viral segments in trans. 21 Among the designs of aforementioned replicons, different viral structural and accessory genes were omitted and different reporter genes were used. In all the cases, S gene was deleted to prevent infectious virus production. Bioluminescence or fluorescence proteins, such as luciferase and GFP, were commonly chosen as reporters to facilitate the detection and standardization of the replicon systems. Furthermore, to select the stable replicon in cells, antibiotic-resistant genes, such as NeoR or BSR, were incorporated into the replicons. 17, 53 In this study, we designed a subgenome of SARS-CoV-2 by omitting structural genes S, E, M, and all the accessory genes except for the 5ʹ and 3ʹ UTR, ORF1ab, and N sequence ( Figure 1 ). This is the minimum version of a replicon that has been proven to work for SARS-CoV-1. 18 We used NLuc as reporter gene to monitor the replicon replication level. A BSR ORF was fused to the NLuc gene with a short linker in between and the fused protein was named Lb. We confirmed that the Lb fusion protein maintains both NLuc and BSR functions ( Figure S1 and data not shown). The N protein is known to be important for the replication of coronaviruses. 32 We thus included the N ORF and its 5ʹ TRS-N sequence downstream of the Lb fusion gene. The full-length replicon cDNA was assembled by in vitro ligation of the synthetic fragments (Figures 1 and 2) , which was subjected to replicon RNA production in vitro and in cells.

First, we took advantage of the pre-engineered T7 promoter at the 5ʹ terminus of F1 to produce IVT mRNA using the IVT-CoV2-Rep cDNA template directly ( Figure 2C,D) . The IVT-CoV2-Rep mRNA rapidly produced NLuc reporter signals upon transfection into host cells, which could be completely abolished by SARS-CoV-2 replication inhibitors. This result suggested that the replicon mRNA is able to initiate replication by at least the production of subgenomic RNAs (Figures 3 and 4) . However, the NLuc signal was rather weak and transitory in various cell lines tested, indicating that the replicon RNA replication is self-limiting or there is a host mechanism restricting the replicon. Considering that the transfected IVT-CoV2-Reported mRNA may act as a pathogen-associated molecular pattern to activate a cellular innate antiviral response, we thus examined whether innate immunity was responsible for the transient replicon replication. While the known deficiency of IFN signaling or response in Huh7.5, Vero, and BHK-21 cells does not support this possibility, [41] [42] [43] [44] [45] [46] we have obtained further evidence that the IVT-CoV2-Rep RNA fails to induce IFN-β in CHO-K1 cells but it attenuates 3p-hpRNA-elicited IFN-β expression ( Figure 5A ). This is consistent with reported findings that SARS-CoV-2 evades type I IFN via antagonizing IFN signaling and response by a set of viral proteins. 38, 40 Furthermore, we have ruled out the potential role of two dsRNAactivated antiviral proteins, specifically PKR and RNase L, in restricting the replicon RNA ( Figure 5C,D) . Taken together, these data suggested that the host cell innate immunity may not be the major cause for the unsustainability of replicon RNA.

The transient replicon RNA replication might be due to the inadequate yield of full-length IVT-CoV2-Rep mRNA for transfection ( Figure S2 , Table S2 ). Therefore, we constructed the DNA-launched BAC-CoV2-Rep replicon, which expresses the full-length replicon mRNA in transfected cells by employing the robust CMV promoter and cellular transcription machinery. Indeed, BAC-CoV2-Rep exhibited a much stronger and sustained NLuc signal than IVT-CoV2-Rep, and addition of the N protein could further enhance the replicon reporter signal in trans, although replicon replication declined over time after it reached the peak level ( Figure 6A ). The N protein expressed from the replicon could also be detected ( Figure 6B ). In addition, the LbN mRNA was detected by Northern blot as a reliable indicator of BAC-CoV2-Rep replication ( Figure 6C ). The antiviral compounds markedly suppressed LbN RNA level by more than 70% ( Figure 7B) , however, the NLuc signal was only slightly inhibited when the treatment started immediately after BAC-CoV2-Rep transfection ( Figure 7A) . A recent study of another pBAC-launched replicon system also reported such resistance to drug treatment but did not provide further explanation. 25 In our study, we showed that a portion of BAC-CoV2-Rep full-length transcripts undergo long-range RNA splicing upstream of Lb ORF, which brings the Lb ORF in close proximity to 5ʹ Cap for Lb translation in a replicon replicationindependent manner (Figure 8 ). We identified three forms of spliced replicon RNA by RT-PCR with primers targeting 5ʹ UTR and Lb sequence ( Figure 8 ). Although we only sequenced a limited number of RT-PCR clones for proof-of-concept in this study, it is expected that additional splicing variants exist in BAC-CoV2-Rep-transfected cells, which can be analyzed further by RNA sequencing. The splicing of replicon RNA transcribed from BAC-CoV2-Rep may also explain the undetectable level of full-length replicon RNA by Northern blot ( Figure 6C ). Interestingly, during the late phase of BAC-CoV2-Rep transfection, the NLuc reporter was more vulnerable to antiviral treatment ( Figure 7C ), which may be due to the dilution, degradation, or epigenetic silencing of the BAC-CoV2-Rep episome as seen in transient DNA transfection systems, 57 or the cellular spliceosome is inhibited by viral NSP16 protein upon expression, 58 therefore, the replicon replication-derived reporter signal becomes predominant in the system. It is worth noting that the observed SARS-CoV-2 RNA splicing is likely specific to the DNA-launched replicon systems, and it should be taken into consideration for its applications in research and development.

While the full-length SARS-CoV-2 cDNA clone exhibited robust viral replication and spread, all the reported SARS-CoV-2 subgenome replicon systems, so far, as well as the replicons described in this study, only supported transient replicon replication. Since our replicons only contain the minimum elements needed for viral genome replication, it is possible that transcomplementing the missing viral proteins may boost and prolong the replicon replication. In line with this, a recently published study, in which only S, E, and M ORF were omitted in their replicon construct, showed a drastic reduction of replication reporter signal after day 2 posttransfection. 19 We have tested cotransfection of IVT-CoV2-Rep mRNA together with different combinations of plasmids expressing viral structural proteins (not S) and accessory proteins, but did not observe any obvious improvement of replicon replication (data not shown). More recently, a SARS-CoV-2 replicon having all viral sequence except for the S ORF also exhibited a decreasing trend of replication after 2 day posttransfection, however, when S was provided in trans, the produced propagation-defective recombinant virus still lost ∼25% infectivity and replication compared to the wild type virus. 22 These results suggest that, in addition to viral proteins, there may be cis elements in S gene and other regions between ORF1b and N gene that could assist a sustained replication of SARS-CoV-2 genome. It is also possible that the engineered reporter and/or antibiotics-resistant gene sequences may negatively affect replicon replication. On a separate note, it has been reported that SARS-CoV-2 NSP1 is a cytotoxic protein that can shut down host cell translation, 59 which may affect the cell viability of replicon-containing cells and eventually halt replicon replication. However, no cytotoxicity was observed in our replicon systems (data not shown), and a reported replicon harboring non-cytopathic NSP1 mutation failed to form stable replicon cell clones. 22 Therefore, to generate a stable SARS-CoV-2 replicon, the architecture of replicon should be further rearranged and optimized to include all necessary genes and cis elements required for viral replication.

Regarding the BAC-CoV2-Rep system, a further optimization effort could be the elimination of RNA splicing through mutating the splicing donor or acceptor sites. By doing this, more full-length replicon mRNA and RTC would be available to subsequent rounds of replication and replication-dependent reporter production.

Furthermore, the BAC-CoV2-Rep holds promise for establishing stable replicon cell lines through blasticidin selection of cells harboring self-replicating replicons. Alternatively, a stable replicating BAC-CoV2-Rep plasmid may be constructed by incorporating the Epstein-Barr virus nuclear antigen-1 its binding oriP DNA sequence, 60 or the replicon DNA cassette can be integrated into host chromosome, by which to serve as a source for stable production of replicon mRNA. In either case, N protein can be stably co-expressed to enhance and prolong the replicon replication. The above proposed work is currently under way in our laboratory.

In summary, we have established two transient minigenome replicons that can be readily applied to research on SARS-CoV-2 replication, virus-host interaction, and antiviral development. This study will also advance the ultimate development of stable SARS-CoV-2 replicon systems amenable to high throughput screening of antiviral drugs to combat the coronavirus pandemic. 

The authors declare no conflict of interests. 

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

http://orcid.org/0000-0001-6194-1742

Haitao Guo http://orcid.org/0000-0002-7146-916X

The peer review history for this article is available at https://publons. com/publon/10.1002/jmv.27650

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Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndromerelated coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

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Mechanisms of alternative pre-messenger RNA splicing

Self-Replicating RNA

Reporter replicons for antiviral drug discovery against positive singlestranded RNA viruses

Coronavirus reverse genetic systems: infectious clones and replicons

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

Mammalian cell transfection: the present and the future

SARS-CoV-2 disrupts splicing, translation, and protein trafficking to suppress host defenses

SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation

Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells

Construction and characterization of two SARS-CoV-2 minigenome replicon systems