key: cord-0923207-sfwxcj6h
authors: Torii, Shiho; Ono, Chikako; Suzuki, Rigel; Morioka, Yuhei; Anzai, Itsuki; Fauzyah, Yuzy; Maeda, Yusuke; Kamitani, Wataru; Fukuhara, Takasuke; Matsuura, Yoshiharu
title: Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction
date: 2020-09-23
journal: bioRxiv
DOI: 10.1101/2020.09.23.309849
sha: 106217d95ee30b8e5dea66ff886954e2c6b7aa73
doc_id: 923207
cord_uid: sfwxcj6h

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). While the development of specific treatments and a vaccine is urgently needed, functional analyses of SARS-CoV-2 have been limited by the lack of convenient mutagenesis methods. In this study, we established a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. Notably, the construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (~5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. We hope that our reverse genetics system will contribute to the further understanding of SARS-CoV-2.

seems difficult to rapidly introduce reporter genes or multiple mutations into viral genes by the classical 48 methods. 49 Recently, a method for the rapid generation of flavivirus infectious clones by circular polymerase 50 extension reaction (CPER) was reported (Edmonds et al., 2013) . In this approach, cDNA fragments 51 covering the full-length viral genome and a linker fragment, which encodes the promoter, polyA signal and 52 ribozyme sequence, are amplified by PCR. Because the amplified fragments are designed to include 53 overlapping ends with adjacent fragments, the amplified fragments can be extended as a circular viral 54 genome with a suitable promoter by an additional PCR using the amplified fragments. By direct 55 transfection of the circular viral genome with the promoter into susceptible cells, infectious viruses can be 56 recovered. This means that infectious clones of flaviviruses can be constructed without any bacterial 57 amplification or in vitro ligation. Using this CPER method, multiple reporter flaviviruses and chimeric 58 2 flaviviruses have been constructed (Tamura et al., 2018; Piyasena et al., 2019) , and a variety of mutant 59 flaviviruses were easily generated and analyzed at the same time (Setoh et al., 2019) . These studies showed 60 that CPER is an effective approach for the characterization of viral proteins. 61 In this study, we tried to establish a CPER method for the construction of SARS-CoV-2 recombinants 62 possessing reporter genes and mutations. In addition, we compared the biological characteristics of the 63 recombinants rescued by the CPER method with the parental SARS-CoV-2. 64 First, we examined whether the CPER approach would be applicable for the construction of an 65 infectious clone of SARS-CoV-2. For this purpose, we used the SARS-CoV-2 strain HuDPKng19-020, subjected to CPER as templates ( Figure 1A and Table S1 ). The cDNA fragments of F9 and F10 were 73 connected to F9/10 before CPER by overlap PCR. A negative control was prepared by CPER using cDNA both Passage 1 (P1) and P2 (data not shown). Infectious titers of the culture supernatants of 89 HEK293-3P6C33 cells at 7 dpt (P0), P1 and P2 viruses, collected at 2 dpi to VeroE6/TMPRSS2 cells, were 90 determined by 50% tissue culture infective dose (TCID50) assays. The infectious titers of the P0, P1, and P2 91 viruses were 10 5.8 , 10 6.3 , and 10 5.8 TCID50/ml, respectively (data not shown), demonstrating that infectious 92 SARS-Co2V-2 was rescued at high titer upon transfection of the CPER product into HEK293-3P6C33 cells, 93 and the recovered viruses were capable of propagating well in VeroE6/TMPRSS2 cells.

To optimize the conditions of recovery of infectious SARS-CoV-2 particles by CPER, the reactions 95 were performed using different numbers of cycles, steps and extension times (conditions 1 to 3 in Star transfected with the CPER product were collected at the indicated time points for 9 days, and infectious 100 titers were determined as the TCID50. In cells transfected with the CPER products without F9/10 (CPER 101 NC), no CPE and no infectious titer in the supernatants was detected until 9 dpt (condition 3 without the 102 expression plasmid of the nucleocapsid protein is shown in Figure S1A ). On the other hand, infectious 103 titers were detected from 5 dpt and reached around 10 7.0 TCID50/ml in the supernatants of cells transfected 104 with the CPER products, regardless of the reaction conditions (condition 3 without the expression plasmid 105 of the nucleocapsid protein is shown in Figure S1A ). No effect of the expression of nucleocapsid protein 106 was observed, suggesting that nucleocapsid is not necessary to recover infectious particles in this method. 107 We selected condition 3 (an initial 2 minutes of denaturation at 98°C; followed by 35 cycles of 10 seconds 108 at 98°C, 15 seconds at 55°C, and 15 minutes at 68°C; and a final extension for 15 minutes at 68°C) for 109 further CPER to generate an infectious cDNA clone for the recovery of infectious particles after 110 transfection into HEK293-3P6C33 cells. Condition 3 was chosen because CPE appeared in cells at 5 dpt of 111 CPER products obtained by condition 3, but at 7 dpt of those obtained by conditions 1 and 2 (data not 112 shown).

To determine the full-length genome sequences of viruses recovered by the CPER method, 2 viruses (#1 114 and #2 in Table S2 ), which were obtained independently at different time points from the supernatants of 115 HEK293-3P6C33 cells, were passaged two times in VeroE6/TMPRSS2 cells, and subjected to Sanger 116 3 sequence analysis with specific primers. Sequence analyses of P0-P2 viruses demonstrated that the 117 recombinant viruses maintained genetic markers (two silent mutations, A7486T and T7489A; Figure 1C ), 118 indicating that there was no contamination of parental virus. Importantly, except for the genetic markers, 119 there was only one difference (T to T/A) in all tested P0 viruses, suggesting that the reverse genetic system 120 for SARS-CoV-2 by CPER had high accuracy. While a large deletion occurred in P1 and P2 of the #2 virus, 121 that deletion was reported to occur during passage in Vero E6 cells (Lau et al., 2020) . 122 Next, we investigated the growth kinetics of recombinant viruses in comparison with parental 123 SARS-CoV-2. Recombinant viruses, which were recovered at 7 dpt of CPER products into 124 HEK293-3P6C33 cells, and parental SARS-CoV-2 were infected into VeroE6/TMPRSS2 cells at RNAs were similar in size ( Figure 1E ). Taken together, these results showed that SARS-CoV-2 rescued by 134 the CPER method exhibits biological characteristics similar to those of the parental virus. HuDPKng19-020), and generated the infectious clone by the CPER method. We confirmed the substitution 173 in the recombinant by Sanger sequence analysis (data not shown), but there was no difference in growth 174 4 kinetics between the WT and D614G recombinant in VeroE6/TMPRSS2 cells ( Figure 2F ). Collectively, 175 these results suggest that our novel reverse genetic system based on CPER is an efficient method for quick 176 generation of recombinants for SARS-CoV-2. 177 Reverse genetics is one of the essential tools to analyze the functions of viral proteins; however, due to 178 the large size of the coronavirus genome, gene manipulations for coronaviruses have been performed by 179 only a very limited number of groups. Here, we established a simple and quick reverse genetics system for 180 SARS-CoV-2 based on the CPER method. The system consists of two steps-namely, amplification of 181 fragments encoding promoter and viral genes, followed by assembly into infectious cDNA clones by PCR 182 without any bacterial amplification. Recombinant viruses were generated with high titers at 7 dpt of CPER 183 products into cells. Using high-fidelity polymerase, SARS-CoV-2 could be rescued with high accuracy, and 184 the rescued virus exhibited characteristics similar to those of the parental virus. It is worth noting that the 185 same tools, i.e., primers and promoter fragments, can be used for generation of viruses possessing multiple 186 mutations or reporters. This method will allow us to conduct high-throughput mutagenesis of SARS-CoV-2, 187 to clarify the function of viral proteins and the mechanisms of propagation and pathogenesis.

In this study, we first report that the CPER method is available to assemble a large size genome (30 kb) To generate avirulent strain for use in developing a safe live-attenuated vaccine for SARS-CoV-2, the 212 CPER method is very useful. To characterize escape mutants occurring through the use of antiviral drugs or 213 vaccinations, CPER can be a robust tool. In comparison with previous reverse genetics systems for 214 coronaviruses, the CPER method is easier and quicker, especially when applied for mutagenesis. We 215 believe that this method will be widely utilized in order to investigate SARS-CoV-2 and further clarify its 

The infectious clones of SARS-CoV-2 carrying sfGFP were also assembled by CPER using 351 pCSII-CoV-2-G10-sfGFP as templates to acquire DNA fragment F10. The HiBiT recombinant 352 SARS-CoV-2 was also generated by CPER as previously described (Tamura et al., 2018) , with some 353 modifications. The HiBiT gene (VSGWRLFKKIS) and a linker sequence (GSSG) were inserted in the N 354 terminus of the ORF6 sequence by overlap PCR using DNA fragment F9 and a specific primer set 355 (ORF6-HiBiT-Rv and ORF6-HiBiT-Fw). Thereafter, CPER was conducted using the resulting fragment F9 356 containing the HiBiT gene. HiBiT luciferase assay 383 SARS-CoV-2 infected cells were collected at the indicated time points and subjected to luciferase assay.

Luciferase activity was measured by using a Nano-Glo HiBiT Lytic assay system (Promega), following the 385 manufacturer's protocols. In brief, the HiBiT assay was conducted by adding Nano-Glo substrate and 

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