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

Takazawa, Shunta; Kotaki, Tomohiro; Nakamura, Satsuki; Utsubo, Chie Aoki; Kameoka, Masanori

doi:10.1016/j.virusres.2023.199176

Cited by 2 publications

(2 citation statements)

References 26 publications

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“…However, the Nluc signal from the more convenient and robust DNA‐launched BAC‐CoV2‐Rep exhibited a high degree of resistance to the above antiviral drugs. Such phenomenon has also been observed in some other plasmid‐vectored SARS‐CoV‐2 replicons, 3–6 indicating the existence of replication‐independent reporter signal in the DNA‐launched replicon systems. Our continuous study revealed that the alternative RNA splicing occurs on the full‐length replicon transcripts derived from the transfected BAC‐CoV2‐Rep template in nucleus, giving rise to spliced RNA variants that carry Nluc ORF in close proximity to 5′ cap of mRNA for direct expression of Nluc independent of replicon replication 2 …”

Section: Introductionmentioning

confidence: 53%

“…The replicon cDNA, under the control of a strong eukaryotic promoter, can be directly transfected into cells without the need of in vitro transcription and continuously supports replicon RNA transcription to maintain replicon replication in the course of transient transfection 1,2 . However, when a reporter gene is engineered into the 3′ portion of replicon genome to monitor replicon replication, the nascent full‐length replicon RNA transcript undergoes mammalian RNA splicing to produce replication‐independent reporter signals, and the reporter signals are variable among different host cell types and from experiment to experiment 2–4,10 (Figure 1). In this study, by using the general host alternative splicing inhibitor Isoginkgetin, we further demonstrated that the host RNA splicing mechanism is responsible for the replicon replication‐independent reporter signal, and inhibition of RNA splicing by Isoginkgetin could highlight the antiviral effect of SARS‐CoV‐2 replication inhibitors when using replicon reporter as a readout (Figures 2–4).…”

Section: Discussionmentioning

confidence: 99%

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Optimization of a DNA‐launched SARS‐CoV‐2 replicon with RNA splicing inhibitor Isoginkgetin

Zhang,

Guo

2024

Journal of Medical Virology

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We have previously developed a bacterial artificial chromosome (BAC)‐vectored SARS‐CoV‐2 replicon, namely BAC‐CoV2‐Rep, which, upon transfection into host cells, serves as a transcription template for SARS‐CoV‐2 replicon mRNA to initiate replicon replication and produce nanoluciferase (Nluc) reporter from the subgenomic viral mRNA. However, an inherent issue of such DNA‐launched replicon system is that the nascent full‐length replicon transcript undergoes process by host RNA splicing machinery, which reduces replicon replication and generates spliced mRNA species expressing NLuc reporter independent of replicon replication. To mitigate this problem, we employed Isoginkgetin, a universal eukaryotic host splicing inhibitor, to treat cells transfected with BAC‐CoV2‐Rep. Isoginkgetin effectively increased the level of full‐length replicon transcripts while concurrently reducing the level of Nluc signal derived from spliced replicon mRNA, making the Nluc reporter signal more correlated with replicon replication, as evidenced by treatment with known SARS‐CoV‐2 replication inhibitors including Remdesivir, GC376, and EIDD‐1931. Thus, our study emphasizes that host RNA splicing is a confounding factor for DNA‐launched SARS‐CoV‐2 replicon systems, which can be mitigated by Isoginkgetin treatment.

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Section: Introductionmentioning

confidence: 53%

Section: Discussionmentioning

confidence: 99%

Optimization of a DNA‐launched SARS‐CoV‐2 replicon with RNA splicing inhibitor Isoginkgetin

Zhang,

Guo

2024

Journal of Medical Virology

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Dimming the corona: studying SARS-coronavirus-2 at reduced biocontainment level using replicons and virus-like particles

Neilsen,

Mathew,

Castro

et al. 2024

mBio

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The coronavirus-induced disease 19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections, has had a devastating impact on millions of lives globally, with severe mortality rates and catastrophic social implications. Developing tools for effective vaccine strategies and platforms is essential for controlling and preventing the recurrence of such pandemics. Moreover, molecular virology tools that facilitate the study of viral pathogens, impact of viral mutations, and interactions with various host proteins are essential. Viral replicon- and virus-like particle (VLP)-based systems are excellent examples of such tools. This review outlines the importance, advantages, and disadvantages of both the replicon- and VLP-based systems that have been developed for SARS-CoV-2 and have helped the scientific community in dimming the intensity of the COVID-19 pandemic.

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

Cited by 2 publications

References 26 publications

Optimization of a DNA‐launched SARS‐CoV‐2 replicon with RNA splicing inhibitor Isoginkgetin

Optimization of a DNA‐launched SARS‐CoV‐2 replicon with RNA splicing inhibitor Isoginkgetin

Dimming the corona: studying SARS-coronavirus-2 at reduced biocontainment level using replicons and virus-like particles

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