Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety level 2 facilities

• Published in: Virus research 2023-09, Vol.334, p.199176-199176, Article 199176
• Main Authors: Takazawa, Shunta, Kotaki, Tomohiro, Nakamura, Satsuki, Utsubo, Chie, Kameoka, Masanori
• Format: Article
• Language: English
• Subjects: Antiviral drug screening; Covid-19; Replication analysis; Replicon; SARS-CoV-2
• ISSN: 0168-1702; 1872-7492
• DOI: 10.1016/j.virusres.2023.199176

•SARS-CoV-2 replicon cDNAs containing HiBiT reporter gene were constructed and cloned into a fosmid vector.•Replication of a series of RNA replicons lacking some open reading frames of SARS-CoV-2 were compared.•Functionalities of SARS-CoV-2 RNA replicons for antiviral drug screening were confirmed.•Replication of SARS-CoV-2 RNA replicons containing puromycin resistant gene showed sustained replication in Huh-7 cells under puromycin pressure. The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has necessitated the global development of countermeasures since its outbreak. However, current therapeutics and vaccines to stop the pandemic are insufficient and this is mainly because of the emergence of resistant variants, which requires the urgent development of new countermeasures, such as antiviral drugs. Replicons, self-replicating RNAs that do not produce virions, are a promising system for this purpose because they safely recreate viral replication, enabling antiviral screening in biosafety level (BSL)-2 facilities. We herein constructed three pCC2Fos-based RNA replicons lacking some open reading frames (ORF) of SARS-CoV-2: the Δorf2–8, Δorf2.4, and Δorf2 replicons, and validated their replication in Huh-7 cells. The functionalities of the Δorf2–8 and Δorf2.4 replicons for antiviral drug screening were also confirmed. We conducted puromycin selection following the construction of the Δorf2.4-puro replicon by inserting a puromycin-resistant gene into the Δorf2.4 replicon. We observed the more sustained replication of the Δorf2.4-puro replicon by puromycin pressure. The present results will contribute to the establishment of a safe and useful replicon system for analyzing SARS-CoV-2 replication mechanisms as well as the development of novel antiviral drugs in BSL-2 facilities.