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Production of a SARS-CoV-2 Virus-Like-Particle System to Investigate Viral Life Cycles In Vitro
Published on: June 6, 2025
Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety
Shunta Takazawa1, Tomohiro Kotaki2, Satsuki Nakamura3
1Department of Public Health, Kobe University Graduate School of Health Sciences, Kobe, Hyogo, Japan; Department of Clinical Laboratory, Kobe City Medical Center General Hospital, Kobe, Hyogo, Japan.
Abstract:
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.
Insights
Researchers developed novel RNA replicons to study SARS-CoV-2 replication safely in BSL-2 facilities. These replicons aid in developing new antiviral drugs against resistant COVID-19 variants.
Area of Science:
- Virology
- Molecular Biology
- Drug Discovery
Background:
- The COVID-19 pandemic, caused by SARS-CoV-2, urgently requires new antiviral drugs due to emerging resistant variants.
- Current vaccines and therapeutics are insufficient to control the pandemic.
- RNA replicons offer a safe method to study viral replication and screen for antivirals in BSL-2 settings.
Purpose of the Study:
- To construct and validate SARS-CoV-2 RNA replicons for antiviral drug screening.
- To establish a safe and efficient system for studying viral replication mechanisms.
- To facilitate the development of novel countermeasures against SARS-CoV-2.
Main Methods:
- Construction of three pCC2Fos-based SARS-CoV-2 RNA replicons (Δorf2-8, Δorf2.4, Δorf2) lacking specific open reading frames.
- Validation of replicon replication in Huh-7 cells.
- Puromycin selection of a modified Δorf2.4 replicon (Δorf2.4-puro) to assess sustained replication under selective pressure.
Main Results:
- Successful construction and replication of the three SARS-CoV-2 RNA replicons in cell culture.
- Demonstrated functionality of Δorf2-8 and Δorf2.4 replicons for antiviral screening.
- Confirmed sustained replication of the Δorf2.4-puro replicon under puromycin pressure.
Conclusions:
- The developed RNA replicons are functional and suitable for antiviral drug screening against SARS-CoV-2.
- This system enables the study of viral replication mechanisms in BSL-2 facilities.
- The findings contribute to the development of new antiviral therapies for COVID-19.
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