Versatile SARS-CoV-2 Reverse-Genetics Systems for the Study of Antiviral Resistance and Replication
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Versatile SARS-CoV-2 Reverse-Genetics Systems for the Study of Antiviral Resistance and Replication. / Fahnøe, Ulrik; Pham, Long V.; Fernandez-Antunez, Carlota; Costa, Rui; Rivera-Rangel, Lizandro René; Galli, Andrea; Feng, Shan; Mikkelsen, Lotte S.; Gottwein, Judith M.; Scheel, Troels K.H.; Ramirez, Santseharay; Bukh, Jens.
In: Viruses, Vol. 14, No. 2, 172, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Versatile SARS-CoV-2 Reverse-Genetics Systems for the Study of Antiviral Resistance and Replication
AU - Fahnøe, Ulrik
AU - Pham, Long V.
AU - Fernandez-Antunez, Carlota
AU - Costa, Rui
AU - Rivera-Rangel, Lizandro René
AU - Galli, Andrea
AU - Feng, Shan
AU - Mikkelsen, Lotte S.
AU - Gottwein, Judith M.
AU - Scheel, Troels K.H.
AU - Ramirez, Santseharay
AU - Bukh, Jens
N1 - Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2022
Y1 - 2022
N2 - The COVID-19 pandemic continues to threaten healthcare systems worldwide due to the limited access to vaccines, suboptimal treatment options, and the continuous emergence of new and more transmissible SARS-CoV-2 variants. Reverse-genetics studies of viral genes and mutations have proven highly valuable in advancing basic virus research, leading to the development of therapeutics. We developed a functional and highly versatile full-length SARS-CoV-2 infectious system by cloning the sequence of a COVID-19 associated virus isolate (DK-AHH1) into a bacterial artificial chromosome (BAC). Viruses recovered after RNA-transfection of in vitro transcripts into Vero E6 cells showed growth kinetics and remdesivir susceptibility similar to the DK-AHH1 virus isolate. Insertion of reporter genes, green fluorescent protein, and nanoluciferase into the ORF7 genomic region led to high levels of reporter activity, which facilitated high throughput treatment experiments. We found that putative coronavirus remdesivir resistance-associated substitutions F480L and V570L—and naturally found polymorphisms A97V, P323L, and N491S, all in nsp12—did not decrease SARS-CoV-2 susceptibility to remdesivir. A nanoluciferase reporter clone with deletion of spike (S), envelope (E), and membrane (M) proteins exhibited high levels of transient replication, was inhibited by remdesivir, and therefore could function as an efficient non-infectious subgenomic replicon system. The developed SARS-CoV-2 reverse-genetics systems, including recombinants to modify infectious viruses and non-infectious subgenomic replicons with autonomous genomic RNA replication, will permit high-throughput cell culture studies—providing fundamental understanding of basic biology of this coronavirus. We have proven the utility of the systems in rapidly introducing mutations in nsp12 and studying their effect on the efficacy of remdesivir, which is used worldwide for the treatment of COVID-19. Our system provides a platform to effectively test the antiviral activity of drugs and the phenotype of SARS-CoV-2 mutants.
AB - The COVID-19 pandemic continues to threaten healthcare systems worldwide due to the limited access to vaccines, suboptimal treatment options, and the continuous emergence of new and more transmissible SARS-CoV-2 variants. Reverse-genetics studies of viral genes and mutations have proven highly valuable in advancing basic virus research, leading to the development of therapeutics. We developed a functional and highly versatile full-length SARS-CoV-2 infectious system by cloning the sequence of a COVID-19 associated virus isolate (DK-AHH1) into a bacterial artificial chromosome (BAC). Viruses recovered after RNA-transfection of in vitro transcripts into Vero E6 cells showed growth kinetics and remdesivir susceptibility similar to the DK-AHH1 virus isolate. Insertion of reporter genes, green fluorescent protein, and nanoluciferase into the ORF7 genomic region led to high levels of reporter activity, which facilitated high throughput treatment experiments. We found that putative coronavirus remdesivir resistance-associated substitutions F480L and V570L—and naturally found polymorphisms A97V, P323L, and N491S, all in nsp12—did not decrease SARS-CoV-2 susceptibility to remdesivir. A nanoluciferase reporter clone with deletion of spike (S), envelope (E), and membrane (M) proteins exhibited high levels of transient replication, was inhibited by remdesivir, and therefore could function as an efficient non-infectious subgenomic replicon system. The developed SARS-CoV-2 reverse-genetics systems, including recombinants to modify infectious viruses and non-infectious subgenomic replicons with autonomous genomic RNA replication, will permit high-throughput cell culture studies—providing fundamental understanding of basic biology of this coronavirus. We have proven the utility of the systems in rapidly introducing mutations in nsp12 and studying their effect on the efficacy of remdesivir, which is used worldwide for the treatment of COVID-19. Our system provides a platform to effectively test the antiviral activity of drugs and the phenotype of SARS-CoV-2 mutants.
KW - GFP
KW - Molecular clone
KW - Nanoluciferase
KW - Polymerase
KW - Remdesivir
KW - Replicon
KW - RNA virus
KW - SARS-CoV-2
U2 - 10.3390/v14020172
DO - 10.3390/v14020172
M3 - Journal article
C2 - 35215765
AN - SCOPUS:85123044181
VL - 14
JO - Viruses
JF - Viruses
SN - 1999-4915
IS - 2
M1 - 172
ER -
ID: 291221685