key: cord-0922702-aj4zg0i2
authors: Tanaka, Tomohisa; Saito, Akatsuki; Suzuki, Tatsuya; Miyamoto, Yoichi; Takayama, Kazuo; Okamoto, Toru; Moriishi, Kohji
title: Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening
date: 2021-12-25
journal: bioRxiv
DOI: 10.1101/2021.12.23.474055
sha: 5ec5bb278a9035e6e4fe6e42aa6192a3ebcc6df9
doc_id: 922702
cord_uid: aj4zg0i2

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-β but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-β. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.

applicable to screening for antiviral compounds targeting SARS-CoV-2 replication, 92 although transient transfection with these vectors or RNAs is still needed for each 93 replicon assay, leading to cumbersome application for studies such as high-throughput 94 screening. Thus, establishment of a cell line harboring stable replication of the SARS- 95 CoV-2 replicon is required for drug screening. 96 In this study, we prepared a BAC vector encoding SARS-CoV-2 reporter 97 replicon RNA with a fusion gene encoding Renilla luciferase and neomycin 98 phosphotransferase II (the replicon-BAC vector), examined the antiviral effects of 99 remdesivir, molnupiravir, type I interferon (IFN), camostat and favipiravir on viral 100 replication in the transient system using the replicon-BAC vector, and established a cell 101 line that stably supports the replication of the replicon. Furthermore, we examined the 102 production of viral factors and the antiviral effect of the reported compounds in the 103 stable replicon cell line. Our stable replicon system for SARS-CoV-2 will be a powerful 104 tool for high-throughput screening for clinically available antivirals. neomycin phosphotransferase (Reo) to establish a stable SARS-CoV-2 replicon cell line. 114 The reporter replicon cDNA was engineered with a BAC due to the large size and to 115 achieve stability of the coronavirus replicase gene (ORF1a, 1b), according to reports 116 described by others (Almazan et al., 2006; Nguyen et al., 2021) . We employed the 117 circular polymerase extension reaction (CPER) method to assemble the BAC vector 118 containing SARS-CoV-2 replicon cDNA (Edmonds et al., 2013; Torii et al., 2021) . 119 Eight fragments covering the region encoding nonstructural polyproteins were 120 subcloned into pCR-blunt (Invitrogen) as templates for fragments F1 to F8 (Fig. 1A) . 121 Fragment F9, which is composed of, in order, a spacer (AscI site), the 3'-end portion of 122 ORF8, transcription-regulating sequence (TRS)-body (TRS-B), the Reo gene, a spacer 123 (BamHI site), the 3'-end portion of ORF8, and TRS-B, was subcloned into pCR-blunt 124 (Fig. 1A , S1A). Fragments F1 to F10, each of which possesses approximately 30-nt 125 overlapping ends for two neighboring fragments, were amplified by PCR using 126 Transient expression of the SARS-CoV-2 reporter replicon in cell culture. 146 We examined whether pBAC-SCoV2-Rep-Reo was active in a transient replicon assay. (Fig. 1C) . Additionally, nsp8, a component of the viral replicase complex, was 6 cells by Northern blotting using a probe specific for the N gene. Three sgRNAs 163 (sgRNA1, 2 and 3, Fig. S1B ) with distinct predicted sizes were detected by Northern 164 blotting (Fig. 1D ). Interestingly, the sgRNA3 level was clearly decreased by treatment 165 with remdesivir but not by treatment with IFN-β in 293T cells (Fig. 1D) . The reduction 166 in the sgRNA3 level by remdesivir treatment was confirmed quantitatively by qRT-167 PCR (Fig. S1E) . The effects of IFN-β and remdesivir on the level of sgRNA3 were well 168 correlated with their effects on the level of N protein in 293T, Huh7 and VeroE6 cells 169 (Fig. 1C, Fig. S1E ). Luciferase activities in 293T, Huh7 and VeroE6 cells transfected replicon with luciferase expression. 190 We attempted to establish a stable replicon cell line using pBAC-SCoV2-Rep-Reo. 191 Because the replicon-BAC vector was designed to confer neomycin resistance to cells, with the vector (data not shown). We established 14 cell clones from these colonies. luciferase activity was observed in clone nos. 1, 3, 4, 6 and 13 than in the other clones 199 ( Fig. S2B ; nos. 1, 3, 4, 6 and 13). However, sgRNA3 was detected in clone no. 3 but not 200 in the other clones (Fig. S2C) . Therefore, clone no. 3 was analyzed further as a stable 201 replicon cell line, designated VeroE6/Rep3. To assess the sustainability of persistent 202 reporter expression in VeroE6/Rep3 cells, we serially passaged the cells in the presence 203 or absence of G418. The luciferase activity in VeroE6/Rep3 cells was maintained for at 204 least 4 weeks in the presence of G418 (Fig. S2D ). The luciferase activity was also 205 maintained but decayed when the cells were cultured for 2 weeks in the absence of 206 G418 (Fig. S2D) . Therefore, it is better to maintain VeroE6/Rep3 cells in the presence 207 of G418, while the medium can also be replaced with G418-free medium during short-208 term assays. 209 To determine whether the replicon was actively replicated in the VeroE6/Rep3 In this study, we established transient and stable replicon systems of SARS-307 CoV-2. The transient replicon system will be available for the comparison of drug 308 efficiency or replication activity among various cell types, while the stable replicon 309 system using VeroE6/Rep3 cells is suggested to be suitable for applications with high-310 throughput screening. Our replicon systems will be helpful to advance knowledge in the 311 field of research on the mechanism of SARS-CoV-2 replication and the development of 312 effective antiviral agents or vaccines for clinical use. covering the replicase complex-encoding region were subcloned into pCR-blunt 454 (Invitrogen) as templates for fragments F1 to F8 (Fig. 1A) . Fragment F9, which is 455 composed of, in order, a spacer (AceI site), the 3'-end portion of ORF8, TRS-B, the Reo 456 gene, a spacer (BamHI site), the 3'-end portion of ORF8, and TRS-B, was subcloned 457 into pCR-blunt (Fig. 1A, S1A) . Eight fragments covering the region encoding the 458 replicase complex (F1-to-F8 in Fig. 1A) were amplified by PCR using the primers listed 459 in Table 1 (Fig. 1A) . Finally, the ten PCR fragments (F1 to F10) were mixed at an The primers for RT-PCR and qRT-PCR are listed in Table 1 . Table 1 and cloned into pBluescriptII. The plasmid was 506 linearized with XhoI and used as the template for probe synthesis. Statistical significance between two groups was determined by Student's t test. 512 Statistical significance for multiple comparisons was determined by one-way ANOVA 513 with Dunnett's post hoc test. A p value less than 0.05 was considered statistically 514 significant (*, p < 0.05; **, p < 0.01; ns, no significance). Post-2 wks without G418

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