TY - JOUR
T1 - A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation
AU - Kotaki, Tomohiro
AU - Xie, Xuping
AU - Shi, Pei Yong
AU - Kameoka, Masanori
N1 - Funding Information:
This work was supported by a young scientist dispatch program, Kobe University: by a subsidy for post-corona society realization, Hyogo prefecture, and by Japan Agency for Medical Research and Development (AMED) under Grant Number JP20he082206. The manuscript was proofread by Enago. We thank Ms. Honoka Yoneda for her technical assistance. We also thank Dr. Masayuki Saijo of the National Institute of Infectious Diseases for providing RNA of SARS-CoV-2 JPN AI/I 004 strain.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12
Y1 - 2021/12
N2 - The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral evaluation. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3000 times higher luminescence than MOCK control cells at 24 h post-electroporation, indicating robust translation and RNA replication of the replicon. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 μM, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 μM). Taken together, this system could be applied to the safe and effective antiviral evaluation without using infectious SARS-CoV-2. Because this is a PCR-based and transient replicon system, further improvement including the establishment of stable cell line must be achieved.
AB - The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral evaluation. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3000 times higher luminescence than MOCK control cells at 24 h post-electroporation, indicating robust translation and RNA replication of the replicon. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 μM, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 μM). Taken together, this system could be applied to the safe and effective antiviral evaluation without using infectious SARS-CoV-2. Because this is a PCR-based and transient replicon system, further improvement including the establishment of stable cell line must be achieved.
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U2 - 10.1038/s41598-021-82055-0
DO - 10.1038/s41598-021-82055-0
M3 - Article
C2 - 33500537
AN - SCOPUS:85099869092
SN - 2045-2322
VL - 11
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 2229
ER -