A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis

Xiaolong Feng; Xiaofan Zhang; Shuangying Jiang; Yuanwei Tang; Chao Cheng; Parthasarathy Abinand Krishna; Xiaoting Wang; Junbiao Dai; Jianyang Zeng; Tian Xia; Dan Zhao

doi:10.1016/j.csbj.2022.08.044

A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis

Comput Struct Biotechnol J. 2022:20:5193-5202. doi: 10.1016/j.csbj.2022.08.044. Epub 2022 Aug 30.

Authors

Xiaolong Feng¹, Xiaofan Zhang^{2

3}, Shuangying Jiang⁴, Yuanwei Tang⁴, Chao Cheng⁵, Parthasarathy Abinand Krishna⁶, Xiaoting Wang⁷, Junbiao Dai⁴, Jianyang Zeng², Tian Xia¹, Dan Zhao²

Affiliations

¹ Department of Pathology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
² Institute for Interdisciplinary Information Sciences, Tsinghua University, Beijing 100084, China.
³ School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 201109, China.
⁴ Guangdong Provincial Key Laboratory of Synthetic Genomics, CAS Key Laboratory of Quantitative Engineering Biology and Shenzhen Key Laboratory of Synthetic Genomics, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
⁵ Institute for Clinical and Translational Research, Baylor College of Medicine, Houston, TX 77030, United States.
⁶ Department of Medicine, Baylor College of Medicine, Houston, TX 77030, United States.
⁷ Silexon AI Technology Co., Ltd., Nanjing, Jiangsu Province 210033, China.

Abstract

The coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seriously affected public health around the world. In-depth studies on the pathogenic mechanisms of SARS-CoV-2 is urgently necessary for pandemic prevention. However, most laboratory studies on SARS-CoV-2 have to be carried out in bio-safety level 3 (BSL-3) laboratories, greatly restricting the progress of relevant experiments. In this study, we used a bacterial artificial chromosome (BAC) method to assemble a SARS-CoV-2 replication and transcription system in Vero E6 cells without virion envelope formation, thus avoiding the risk of coronavirus exposure. Furthermore, an improved real-time quantitative reverse transcription PCR (RT-qPCR) approach was used to distinguish the replication of full-length replicon RNAs and transcription of subgenomic RNAs (sgRNAs). Using the SARS-CoV-2 replicon, we demonstrated that the nucleocapsid (N) protein of SARS-CoV-2 facilitates the transcription of sgRNAs in the discontinuous synthesis process. Moreover, two high-frequency mutants of N protein, R203K and S194L, can obviously enhance the transcription level of the replicon, hinting that these mutations likely allow SARS-CoV-2 to spread and reproduce more quickly. In addition, remdesivir and chloroquine, two well-known drugs demonstrated to be effective against coronavirus in previous studies, also inhibited the transcription of our replicon, indicating the potential applications of this system in antiviral drug discovery. Overall, we developed a bio-safe and valuable replicon system of SARS-CoV-2 that is useful to study the mechanisms of viral RNA synthesis and has potential in novel antiviral drug screening.

Keywords: Antiviral drug; BAC, bacterial artificial chromosome; BSL-3, bio-safety level 3; Bacterial artificial chromosome; CMV, cytomegalovirus; COVID-19, coronavirus disease-2019; E, envelop; EGFP, enhanced green fluorescent protein; HDV, hepatitis delta virus; HTS, high-throughput screening; M, membrane; N, nucleocapsid; Nucleocapsid protein; ORF, open reading frame; RNP, ribonucleoprotein; RTCs, replication-transcription complexes; Replicon; Rz, ribozyme; S, spike; SARS-CoV-2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SNPs; SNPs, single nucleotide polymorphisms; SR, serine/arginine; TRSs, transcription-regulatory sequences; YCp, yeast centromere plasmid; gRNA, genomic RNA; nsps, non-structural proteins; sgRNAs, subgenomic RNAs; sgmRNAs, subgenomic mRNAs.