bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.294330. this version posted September 12, 2020.

The copyright holder for this

1 A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of

2 SARS-CoV-2

* *
4 Yang Zhang1, Wuhui Song1, Shuiye Chen1, Zhenghong Yuan1 , Zhigang Yi1,2

6 1. Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic

7 Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, 200032, PR

8 China

9 2. Shanghai public health clinical center, Fudan University, Shanghai, 201508, PR China

10 Runing title: A replicon of SARS-CoV-2

11

12

13 * Contact authors: Zhigang Yi, Shanghai Medical College, Fudan University, Shanghai

14 200032, China, Email: zgyi@fudan.edu.cn. 138 YiXueYuan Road, Shanghai 200032, China.

15 Zhenghong Yuan, Shanghai Medical College, Fudan University, Shanghai 200032, China

16 Email: zhyuan@shmu.edu. 138 YiXueYuan Road, Shanghai 200032, China. Tel:

17 +86-21-54237669. Fax: +86-21-64227201

18

19

20

21

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22 Abstract

23 Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To

24 facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety

25 level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of

26 SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded

27 in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes

28 spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated

29 in various cell lines, and was sensitive to interferon alpha (IFN-α), remdesivir, but was

30 resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon

31 was also sensitive overexpression of zinc-finger antiviral protein (ZAP). We also constructed

32 a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes

33 it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This

34 replicon system would be helpful for performing antiviral screening and dissecting virus-host

35 interactions.

36

37

38

39

2
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40 Introduction

41 The pandemic COVID-19 has infected over 26 million people and caused over 800,000

42 mortalities. (https://www.who.int/emergencies/diseases/novel-coronavirus-2019). It is caused

1-3
43 by infection with a novel beta coronavirus SARS-CoV-2 . Vaccines and antiviral agents

44 are in urgent need to stop the pandemic. Despite great progresses on SARS-CoV-2 vaccine

45 development and clinical trails 4, the protection efficacy of the vaccines still remains to be

46 determined. There have been trials of antiviral agents such as remdesivir and chloroquine for

5-7
47 COVID-19 treatment, however, efficacy of these antiviral agents remains uncertainty .

48 Development of convenient tools for antiviral screening will speed up seeking effective

49 antiviral agents against SARS-CoV-2. Recently, infectious clones of SARS-CoV-2 with

50 reporter genes 8, 9 provide elegant tools for antiviral development. However, due to the safety

51 issue and requirement for biosafety level 3 laboratory, usage of these infectious clones is

52 limited. Non-infectious replicon system that recapitulates authentic viral replication without

53 virion production can be used to perform screening for antivirals that target viral replication

54 process.

55 SARS-CoV-2 contains an approximate 29kb, single stranded, positive sense RNA

56 genome. About two-thirds of the viral genome encodes open reading frames (ORFs) for

57 translation of the replicase and transcriptase proteins, the only ORFs translated from the viral

58 genome. The translated replicase and transcriptase proteins engage viral genome to assemble

59 the replicase-transcriptase complex on endoplasmic reticulum membrane, forming a

60 membranous compartment. Within the membranous compartment, replicase-transcriptase

3
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61 complex initiates viral replication and transcription. Transcription of the 3'-most third

62 genomes by viral replicase-transcriptase generates various subgenomic mRNAs that encode

10
63 structural proteins and accessory genes . Structural proteins include the spike (S),

64 membrane (M), envelop (E) proteins and nucleocapsid (N) participate in virion assembly 10.

65 In this study, we generated a replicon system for SARS-CoV-2, nCoV-SH01 strain with

66 secreted Gaussia luciferase (sGluc) as a reporter gene. The cDNA of viral genome with

67 deletion of S, M, E genes was cloned into a bacterial artificial chromosome (BAC) vector.

68 The reporter gene sGluc was encoded in subgenomic viral RNA. The viral RNA was

69 transcribed in vitro by T7 polymerase. Upon transfection into cells, the viral replication was

70 detected, as evidenced by expression of subgenomic viral RNA-encoded sGluc. The viral

71 replication was sensitive to interferon alpha (IFN-α), remdesivir, but was resistant to hepatitis

72 C virus inhibitors daclatasvir and sofosbuvir. The replicon genomes could also be assembled

73 by in-vitro-ligation of four DNA fragments and the RNA generated by the in-vitro-ligated

74 DNA template was capable of replication as the RNAs derived from the BAC-template. Thus,

75 we provided a simple SARS-CoV-2 replicon system for antiviral development.

76 Results

77 Construction of a bacterial artificial chromosome (BAC) based SARS-CoV-2 replicon.

78 Total RNAs were extracted from SARS-CoV-2 (nCoV-SH01) infected cells 11, and then

79 reversely transcribed by superscript IV with random primer. Totally, 20 fragments with

80 approximate l.5kb-length encompassing the whole viral genomes were amplified with

81 specific primers according to the illumina-sequenced viral genome (MT121215), cloned and

4
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82 sequenced. The fragments were then assembled by fusion PCR and subcloning into larger

83 fragments A (1-8586nt)，B (8587-15102nt), C (15103-21562nt) and D and cloned into a

84 homemade cloning vector pLC-Zero-blunt (Fig.1). Took a similar strategy for construction of

85 SARS-CoV replicon 12, we deleted the structural protein genes and retained the N gene and

86 essential promoter regions. We replaced the S gene region with a reporter gene cassette,

87 including secreted Gaussia luciferase (sGluc), foot-and-mouth disease virus (FMDV) 2A

88 peptide and blasticidin (BSD), whose expression was driven by the promoter of S gene in the

89 subgenomic mRNA (Fig. 1). To facilitate cloning, a BamHI site was introduced downstream

90 the genome position of 21562 (nt) in the pLC-nCoV-C plasmid. T7 promoter was added

91 before the 5' viral genome in the fragment A for in vitro transcription with T7 polymerse. The

92 3' viral genome was flanked with polyA30, hepatitis delta virus ribozymes (HDVr) and

93 terminator sequence for T7 polymerase (T7T) (Fig. 1), which facilitates in-vitro transcription

94 without linearization and production of precise polyadenylated viral RNA. The fragments of

95 A, B, C and D were assembled further into AB and CD in pLC-Zero-blunt and then

96 sequentially cloned into a modified BAC vector to get the final plasmid

97 pBAC-sgnCoV-sGluc (Fig. 1).

98 Replication of SARS-CoV-2 replicon in cells

99 The plasmid pBAC-sgnCoV-sGluc was used directly as template for in-vitro

100 transcription to produce 5'-capped replicon RNA. Replicon RNA was then co-transfected

101 with N mRNA into various cell lines. RNA replication was monitored by measuring the

102 secreted Gaussia luciferase activity in the supernatants. Enzymatic dead mutants

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103 (759-SAA-761) of the RNA dependent RNA polymerase nsp1213 were introduced and the

104 mutated replicon served as a non-replication control. As expected, SAA RNA did not

105 replicate, without increase of luciferase activity in the transfected Huh7, Huh7.5, Vero and

106 BHK-21 cells. In contrast, transfection of wild type (WT) replicon RNA resulted in obvious

107 increase of luciferase activity (Fig. 2a, b, c, d), indicating active viral replication. Huh7.5 cell

108 is a subclone of Huh7 cells with deficiency in RIG-I and MDA-5 signaling14, 15. Vero cell is

109 routinely used for SARS-CoV-2 isolation. Notably, replicon replication was less efficient in

110 Huh7.5 and Vero cells (Fig. 2b and c) whereas robust in BHK-21 cells (Fig. 2d), suggesting

111 that cellular environment may regulate SARS-CoV-2 replication. In consistent with previous

112 studies, co-transfection of N mRNA enhanced viral replication (Fig. 2a, b, c, d), which is

113 probably due to the suppression of innate immune response16. For convenience, we tried to

114 establish an N-expressed cell line (Fig.2e). Compared with GFP-expressed cells, Huh7 cells

115 expressing N supported more robust viral replication (Fig. 2f).

116 Sensitivity of the SARS-CoV-2 replicon to antiviral agents

117 We tested the sensitivity of SARS-CoV-2 replicon to remdesivir, which has been

118 demonstrated to inhibit SARS-CoV-2 viral infection17. Huh7 cells were first treated with

119 remdesivir at various concentrations (10µm, 3.7µm, 1µm, 100nm, 10nm) as reported17, and

120 then the cells were co-transfected with replicon RNA (WT or SAA) and N mRNA. The

121 luciferase activity in the supernatants was measured at various time points after transfection.

122 We found that at all concentrations, remdesivir effectively inhibited replicon replication to a

123 similar level as SAA (Fig.3a). We also examined if the remdesivir inhibited established viral

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124 RNA replication. We first transfected replicon RNA, and then added remdesivir eight hours

125 post transfection and monitored viral replication at various time points post treatment. Under

126 this condition, remdesivir also reduced whereas did not completely block viral replication

127 (Fig. 3b). Then we tested the sensitivity of SARS-CoV-2 replicon to other antiviral agents.

128 Huh7 cells were first treated with interferon ahpha (IFN-α)(100U/ml), remdesivir (10 nM),

129 daclatasvir (1 µM), sofosbuvir (10 µM) and 2’-C-Methylcytidine (2CMC)(50 µM) for four

130 hours, then the cells were co-transfected with replicon RNA (WT or SAA) and N mRNA.

131 The luciferase activity in the supernatants was measured at the various time points after

132 transfection. IFN-α and remdesivir have been demonstrated to inhibit SARS-CoV-2 viral

133 infection17, 18. Daclatasvir and sofosbuvir are direct antivirals targeting hepatitis C virus

134 NS5A19 and RNA dependent RNA polymerase20, respectively. 2’-C-Methylcytidine is a

21
135 nucleoside inhibitor of HCV NS5B polymerase . As shown in Figure 3c, IFN-α and

136 remdesivir effectively inhibited sgnCoV-sGluc replication. Notably, IFN-α started to reduce

137 the reporter gene expression at early time point (8 hours post transfection), manifested as

138 lower luciferase activity then the SAA mutant, which suggests that IFN-α may block

139 translation of the viral subgenomic mRNA. Remdesivir effectively inhibited the luciferase

140 expression to a similar level of SAA. In contrast, sofosbuvir and 2’-C-Methylcytidine hardly

141 reduced luciferase expression and daclatasvir had no effect on luciferase expression (Fig. 3c).

142 These results demonstrate that SARS-CoV-2 replicon is sensitive to antiviral agents against

143 SARS-CoV-2.

144 Sensitivity of SARS-CoV-2 replicon to overexpression of Zinc-finger antiviral protein

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145 (ZAP).

146 Zinc-finger antiviral protein recognizes CpG dinucleotide on non-self RNA and exerts

147 antiviral activity22. There is extreme low CpG content of SARS-CoV-2 genome, suggesting

148 SARS-CoV-2 may evolve under the pressure of ZAP23, 24. We generated a stable Huh7 cell

149 line expressing the long isoform of ZAP (ZAPL) and examined the replicon RNA replication

150 in the Huh7-ZAPL cells (Fig. 4a). There was about 10-fold reduction of replicon replication

151 in Huh7-ZAPL comparing with that in GFP expressing cells (Huh-GFP) (Fig. 4b), suggesting

152 replicon replication is sensitive to ZAPL overexpression.

153 Assembly of SARS-CoV-2 replicon by in vitro ligation.

154 As the difficulties to manipulate with BAC vectors, we tried to assemble the four

155 fragments A, B, C and D by in-vitro ligation. We introduced additional BsaI sites into the 5'

156 and 3' of each fragment in the assembly plasmids pLC-nCoV-A, pLC-nCoV-B, pLC-nCoV-C,

157 pnCoV-sGluc, retaining all the original restrictions enzymes. The fragments were released

158 from the plasmid by BsaI digestion, and assembled by in-vitro ligation with T4 ligase (Fig.

159 5a). RNAs transcribed from the in-vitro ligated template replicated similar as the RNAs

160 transcribed from BAC vector (Fig. 5b).

161 Discussion

162 In this study, we described a replicon system of SARS-CoV-2. In the replicon, we

163 deleted the spike (S), membrane (M), envelop (E) genes that are essential for virion

164 production, making it non-infectious and safe (Fig. 1). Upon transfection into various cells,

165 the replicon RNA could replicate, manifested by the expression of subgenomic mRNA

8
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166 encoded sGluc (Fig. 2). The viral replication was inhibited by anti-SARS-CoV-2 antiviral

167 agent remdesivir and by IFN-α but was not by antivirals against hepatitis C virus (Fig. 3).

168 This replicon system avoids requirement for specific biosafety facilities. The BAC-vectored

169 replicon system does not need in vitro ligation or recombination in yeast, which simplifies the

170 experiment processes. Thus this replicon system would be used conveniently to perform

171 antiviral screening against SARS-CoV-2. We also constructed a four-plasmid in-vitro ligation

172 system that is compatible with the BAC system. Replicon RNAs produced from the in-vitro

173 ligated replicate similarly with the RNAs transcribed from BAC plasmids (Fig. 5). It is easy

174 to introduce desired mutations into the assembly plasmids for in-vitro ligation, which make it

175 suitable for dissecting the effect of emerging mutations on viral replication and molecular

176 mechanisms of viral replication.

177 Material and methods

178 Cloning

179 Total RNAs were extracted from SARS-CoV-2 (nCoV-SH01) infected cells 11, reversely

180 transcribed by superscript IV (Invitrogen) with random primer. Totally 20 fragments with

181 approximate l.5kb-length encompassing the whole viral genomes were amplified with

182 specific primers according to the illumina-sequenced viral genome (MT121215), cloned into

183 a homemade cloning vector pZero-blunt and sequenced. Four larger fragments A (1-8586nt)，

184 B (8587-15102nt, C (15103-21562nt) and D with deletion of structural protein genes and

185 addition of reporter gene cassette were assembled by fusion PCR and subcloning, and then

186 cloned into a homemade cloning vector pLC-Zero-blunt and pcDNA3.1 (invitrogen),

9
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187 respectively, resulted in plasmids pLC-nCoV-A, pLC-nCoV-B, pLC-nCoV-C, and

188 pnCoV-D-sGluc. To facilitate cloning, a BamHI site was introduced downstream the genome

189 position of 21562 (nt) in the plasmid pLC-nCoV-C. In fragment A, T7 promoter was added

190 before the 5' viral genome. In fragment D, a expression cassette containing secreted Gaussia

191 luciferase (sGluc), foot-and-mouth disease virus (FMDV) 2A peptide (NFDLL KLAGD

192 VESNP GP) and blasticidin (BSD) was added upstream the 5'-postion of viral genome. The 3'

193 viral genome was flanked with polyA30, hepatitis delta virus ribozymes (HDVr) and

194 terminator sequence for T7 polymerase (T7T). Inactive mutants (759-SAA-761) of the RNA

195 dependent RNA polymerase nsp12 was introduced into the C fragment at the predicted

196 catalytic residues (759-SDD-761)13 by fusion PCR mediated mutagenesis.

197 To assemble the four fragments into bacterial artificial chromosome (BAC) vector, first

198 we modified the pSMART-BAC v2.0 (Lucigen) to get ride of unwanted restriction enzymes

199 and added AatII and XhoI sites to facilitate cloning by multiple rounds of fusion-PCR

200 mediated mutagenesis. The fragments were then sequentially cloned into the BAC vector. We

201 first assemble the fragment A and B, C and D by enzyme digestion to get the plasmid

202 pLC-nCoV-AB and pLC-nCoV-CD, respectively. Then the AB fragments were cloned into

203 the SbfI/XhoI site to generate pBAC-sgnCoV-AB. Then the CD fragments were ligated into

204 the SacI/AsisI site to get pBAC-sgnCoV-sGluc. BAC plasmid was delivered into

205 BAC-Optimized Replicator v2.0 Electrocompetent Cells (Lucigen) by electroporation and

206 bacteria was propagated according to the manufacturer’s guide. Colonies were picked and

207 cultured in LB medium containing 12.5 µg/ml chloramphenicol. L-arabinose was added to

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208 cultures when the OD600 reaches 0.2-0.3 to increase the plasmid copy numbers.

209 For assembly of SARS-CoV-2 replicon by in-vitro-ligation, we first got rid of the BsaI

210 site on fragment C by fusion-PCR mediated synonymous mutagenesis. The BsaI sites were

211 added into the 5' and 3' of the fragment A, B, C and D in the plasmids of pLC-nCoV-A,

212 pLC-nCoV-B, pLC-nCoV-C, and pnCoV-D-sGluc by fusion PCR-mediated cloning, resulted

213 in the plasmids pLC-nCoV-A-BsaI, pLC-nCoV-B-BsaI, pLC-nCoV-C-BsaI, and

214 pnCoV-D-sGluc-BsaI. The plasmids retained all the original enzymatic sites, for convenience

215 to swap into the BAC vector if desired. The fragments were released from the plasmids by

216 BsaI digestion, after gel purification and ligated by T4 ligase.

217 To construct lentiviral vector expression plasmids, sequences encoding the GFP,

218 SARS-CoV-2 nucleocapsid protein (N) were cloned into the XbaI/BsrGI site of

219 pTRIP-IRES-BSD. Sequence encoding the long isoform of Zinc-finger antiviral protein

220 (ZAPL) were synthesized by Wuxi Qinglan Biotech (Wuxi, China) and cloned into the

221 XbaI/BamHI site of pTRIP-IRES-BSD. An HA tag was added into the N-terminal of ZAP.

222 For production of N mRNA, sequence encoding N was first cloned into the KpnI/BamHI

223 sites of phCMV to get the plasmid phCMV-N. All the plasmids were verified by Sanger

224 sequencing. The detail information was available upon request.

225 Cell lines

226 The human hepatoma cells Huh 7, baby hamster kidney cells BKH-21, Vero E6 cells were

227 purchased from the Cell Bank of the Chinese Academy of Sciences (www.cellbank.org.cn)

228 and routinely maintained in Dulbecco’s modified medium supplemented with 10 % FBS

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229 (Gibco) and 25 mM HEPES (Gibco). Huh 7.5 (Kindly provided by C. Rice) cells were

230 routinely maintained in a similar medium supplemented with non-essential amino acids

231 (Gibco). Huh7-GFP, Huh7-N, Huh7-ZAPL cell line was routinely maintained in the medium

232 supplemented with 0.5 µg/ml blasticidin.

233 Lentivirus pseudoparticle

234 VSV-G-pseudotyped lentiviral particles were prepared by co-transfection of VSV-G, HIV

235 gag-pol and lentiviral provirus plasmids into HEK293T cells. The medium overlying the cells

236 was harvested at 48 h after transfection, filtered through a 0.45-µm filter, and stored at -80°C.

237 Cells were transduced with the pseudoparticles in the presence of 8 µg/ml Polybrene.

238 Inhibitors

239 Remdesivir (GS-5734), Daclatasvir (S1482)，Sofosbuvir (GS-7977) were purchased from

240 Selleckchem, 2’-C-Methylcytidine (HY-10468) was purchased from MedChem Express,

241 IFN-α (11200-2) was purchased from PBL.

242 Antibodies

243 Anti-β-actin antibody (Sigma; A1978) was used at 1:5000 dilution; Anti-HA antibody (CST;

244 37243) was used at 1:000 dilution; Anti-GFP antibody (Santa Cruz;sc-9996) was used at

245 1:1000 dilution; Anti-ZAPL antibody (Proteintech; 16820-1-AP) was used at 1:1000 dilution;

246 Anti-N antibody(GeneTex; GTX632269) was used at 1:500 dilution; Goat-anti-mouse IRDye

247 800CW secondary antibody (licor; 926-32210) was used at 1:10,000 dilution.

248 Goat-anti-rabbit IRDye 800CW secondary antibody (licor; 926-32211) was used at 1:10,000

249 dilution in western blotting.

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250 Western blotting

251 After washing with PBS, cells were lysed with 2 × SDS loading buffer (100 mM Tris-Cl [pH

252 6.8], 4% SDS, 0.2% bromophenol blue, 20% glycerol, 10% 2-mercaptoethanol) and then

253 boiled for 5 min. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose

254 membrane. The membranes were incubated with blocking buffer (PBS, 5% milk, 0.05%

255 Tween) for 1 h and then with primary antibody diluted in the blocking buffer. After three

256 washes with PBST (PBS, 0.05% Tween), the membranes were incubated with secondary

257 antibody. After three washes with PBST, the membrane was visualized by Western Lightning

258 Plus-ECL substrate (PerkinElmer, NEL10500) or by Odyssey CLx Imaging System.

259 In-vitro ligation

260 BsaI digested fragment were gel purified by using Gel Extraction Kit (OMEGA) and ligated

261 with T4 ligase (New England Biolabs) at room temperature for 1h. The ligation products

262 were phenol/chloroform extracted, precipitated by absolute ethanol, and resuspended in

263 nuclease-free water, quantified by determining the A260 absorbance.

264 In-vitro transcription

265 BAC-based sgnCoV-sGluc plasmids or purified in-vitro ligated products were used as

266 templates for the in-vitro transcription by mMESSAGE mMACHINE T7 Transcription Kit

267 (Ambion) according to the manufacturer's protocol. For N mRNA production, we amplified

268 the N coding region by PCR (sense: GGC ACA CCC CTT TGG CTC T; antisense: TTT TTT

269 TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TCT AGG CCT GAG TTG AGT CAG

270 CAC) with phCMV-N as template. Then the purified PCR product was used as template for

13
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271 in-vitro transcription by mMESSAGE mMACHINE T7 Transcription Kit as described above.

272 RNA was purified by RNeasy mini Elute (Qiagen) and eluted in nuclease-free water,

273 quantified by determining the A260 absorbance.

274 Transfection

275 Cells were seeding onto 48-well plates at a density of 7.5×104 per well and then transfected

276 with 0.3 µg in-vitro-transcribed RNA using a TransIT-mRNA transfection kit (Mirus)

277 according to the manufacturer’s protocol.

278 Luciferase activity

279 Supernatants were taken from cell medium and mixed with equal volume of 2 ×lysis buffer

280 (Promega). Luciferase activity was measured with Renilla luciferase substrate (Promega)

281 according to the manufacturer's protocol.

282 Acknowledgements

283 This work was supported in part by the National Science and Technology Major Project of

284 China (2017ZX10103009), Key Emergency Project of Shanghai Science and Technology

285 Committee (20411950103). The funders had no role in study design, data collection and

286 analysis, decision to publish, or preparation of the manuscript.

287 Author Contributions

288 Conceived the study: Z Yi; conducted the study: Y Zhang, W Song, S Chen, Z Yi; Data

289 analysis: Z Yi, Y Zhang; Manuscript draft: Y Zhang, Z Yi; Resources: Z Yuan, Z Yi

290 Conflict of Interest

291 The authors declare no conflict of interest.

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327 16. Ye, Y., Hauns, K., Langland, J.O., Jacobs, B.L. & Hogue, B.G. Mouse hepatitis

328 coronavirus A59 nucleocapsid protein is a type I interferon antagonist. J Virol 81,

329 2554-2563 (2007).

330 17. Wang, M. et al. Remdesivir and chloroquine effectively inhibit the recently emerged

331 novel coronavirus (2019-nCoV) in vitro. Cell research 30, 269-271 (2020).

332 18. Li, L. et al. Antiviral Agent Therapy Optimization in Special Populations of

333 COVID-19 Patients. Drug design, development and therapy 14, 3001-3013 (2020).

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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

334 19. Gao, M. et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a

335 potent clinical effect. Nature 465, 96-100 (2010).

336 20. Kohli, A., Shaffer, A., Sherman, A. & Kottilil, S. Treatment of hepatitis C: a

337 systematic review. Jama 312, 631-640 (2014).

338 21. Mathy, J.E., Ma, S., Compton, T. & Lin, K. Combinations of cyclophilin inhibitor

339 NIM811 with hepatitis C Virus NS3-4A Protease or NS5B polymerase inhibitors

340 enhance antiviral activity and suppress the emergence of resistance. Antimicrobial

341 agents and chemotherapy 52, 3267-3275 (2008).

342 22. Takata, M.A. et al. CG dinucleotide suppression enables antiviral defence targeting

343 non-self RNA. Nature 550, 124-127 (2017).

344 23. Zhang, J. et al. Multi-site co-mutations and 5’UTR CpG immunity escape drive the

345 evolution of SARS-CoV-2. bioRxiv, 2020.2007.2021.213405 (2020).

346 24. Xia, X. Extreme Genomic CpG Deficiency in SARS-CoV-2 and Evasion of Host

347 Antiviral Defense. Molecular biology and evolution 37, 2699-2705 (2020).

348

349

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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

350 Figure Legends

351 Fig. 1. Schematic of construction of BAC-based replicon of SARS-CoV-2. Twenty

352 fragments encompassing the whole viral genomes were amplified, cloned and sequenced.

353 Four larger fragments A (1-8586nt)，B (8587-15102nt, C (15103-21562nt) and D with

354 deletion of structural protein genes and addition of reporter gene cassette were assembled and

355 cloned. To facilitate cloning, a BamHI site (in bold) was introduced downstream the genome

356 position of 21562 (nt). In fragment A, T7 promoter (T7P) was added. In fragment D, an

357 expression cassette containing secreted Gaussia luciferase (sGluc), foot-and-mouth disease

358 virus (FMDV) 2A peptide and blasticidin (BSD) was added. The 3' viral genome was flanked

359 with polyA30, hepatitis delta virus ribozymes (HDVr) and terminator sequence for T7

360 polymerase (T7T). Then the fragments were assembled and sequentially cloned into a

361 modified BAC plasmid. Upon transfected into cells, the replicon RNA can be used as

362 template for RNA replication or transcription to produce subgenomic RNA. The sGluc

363 subgenomic RNA is translated to produce sGluc.

364 Fig. 2. Replication of sgnCoV-sGluc in different cells. a-d Huh7, Huh7.5, Vero and

365 BHK-21 cells were transfected with in-vitro-transcribed replicon RNA(WT) or the nsp12

366 polymerase active-site mutant (SAA). An mRNA encoding the SARS-CoV-2 N protein was

367 co-transfected or not. The luciferase activity in the supernatants was measured at the time

368 points indicated. Medium was changed at each time point. Data are shown as mean±SD (n=3).

369 e-f Replication of replicon RNA in Huh7 cells overexpressed N protein. e Huh7 cells

370 overexpressed GFP protein or N protein were analyzed by Western blotting with the

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 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.294330. this version posted September 12, 2020. The copyright holder for this
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371 indicated antibodies. f Huh7-GFP and Huh7-N cells were transfected with replicon RNA

372 (WT or SAA). The luciferase activity in the supernatants was measured at the time points

373 indicated. Medium was changed at 8 hours post transfection. Data are shown as mean±SD

374 (n=3).

375 Fig. 3. Sensitivity of the SARS-CoV-2 replicon to antiviral agents. a Huh7 cells were

376 treated with remdesivir as the indicated concentration. Four hours later, cells were

377 co-transfected with replicon RNA (WT or SAA) and N mRNA. The luciferase activity in the

378 supernatants was measured at the time points indicated. b Huh7 cells were co-transfected

379 with replicon RNA (WT or SAA) and N mRNA. Eight hours later, medium was changed

380 with remdesivir as the indicated concentration. The luciferase activity in the supernatants was

381 measured at the time points indicated. c Huh7 cells were treated with remdesivir (10nM),

382 IFN-α(100 U/ml), daclatasvir(1 µM), sofosbuvir(10 µM), 2CMC(50 µM). Four hours later,

383 cells were co-transfected with replicon RNA (WT or SAA) and N mRNA. The luciferase

384 activity in the supernatants was measured at the time points indicated. Medium was changed

385 at 8 hours post transfection. Data are shown as mean±SD (n=3).

386 Fig. 4. Sensitivity of SARS-CoV-2 replicon to overexpression of Zinc-finger

387 antiviral protein (ZAP). a Huh7 cells overexpressed ZAPL protein or GFP protein were

388 analyzed by Western blotting with the indicated antibodies. b Huh7-GFP and Huh7-ZAPL

389 cells were co-transfected with replicon RNA (WT or SAA) and N mRNA. The luciferase

390 activity in the supernatants was measured at the time points indicated. Medium was changed

391 at 8 hours post transfection. Data are shown as mean±SD (n=3).

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 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.294330. this version posted September 12, 2020. The copyright holder for this
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

392 Fig. 5. Assembly of SARS-CoV-2 replicon by in vitro ligation. a Schematic of the

393 in-vitro ligation system for SARS-CoV-2 replicon. b Huh7 cells were co-transfected with

394 replicon RNA (WT or SAA) and N mRNA generated by BAC-based system or in-vitro

395 ligation system. The luciferase activity in the supernatants was measured at the time points

396 indicated. Medium was changed at 8 hours post transfection. Data are shown as mean±SD

397 (n=3).

398

399

400

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401 Figure 1

402
403

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404 Figure 2

405
406

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 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.294330. this version posted September 12, 2020. The copyright holder for this
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407 Figure 3

408
409

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 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.294330. this version posted September 12, 2020. The copyright holder for this
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410 Figure 4

411
412

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 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.294330. this version posted September 12, 2020. The copyright holder for this
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413 Figure 5

414

415

416

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