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bioRxiv · 10.1101/2024.07.28.605443

A detailed protocol for generating spike trans-complemented SARS-CoV-2 replicons

Yang, Q.·Manicassamy, B.·Neelamegham, S.
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Abstract

Multiple approaches have been implemented for basic science studies that attempt to investigate SARS-CoV-2 biology or virology in biosafety level-2 setting. These include pseudotyped-virus based on lentivirus and vesicular stomatitis virus, virus-like particles that only contain the SARS-CoV-2 structural proteins, and single-cycle replicons. Among these, the single-cycle replicons most closely resemble the authentic virus as they essentially include the full viral genome, except for essential elements required for active virus multiplication. In this regard, we previously developed a SARS-CoV-2 replicon system where a Gaussia Dura luciferase-P2A-mNeonGreen reporter cassette replaced viral spike. In this short paper, we present an optimized protocol for the use of this reagent that overcomes previous technical limitations. We demonstrate that co-transfection of this bacmid along with spike plasmid, using the improved protocol, yields high-quality spike bearing SARS-CoV-2 virus particles with single-cycle infectivity. Due to the nature of bacmid construction, this approach is particularly useful for studying the impact of spike mutagenesis on virus evolution in BSL-2 setting.

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Yang, Q., Manicassamy, B., Neelamegham, S.. 2024-07-30. A detailed protocol for generating spike trans-complemented SARS-CoV-2 replicons. https://doi.org/10.1101/2024.07.28.605443

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