Abstract
INTRODUCTION: The large size of coronavirus genome, along with the instability of certain genomic sequences, makes the construction of reverse genetics for coronaviruses particularly challenging. The rapid development and application of reverse genetics systems for coronaviruses require further exploration. METHODS: Using transformation-associated recombination (TAR) cloning in yeast and the in vitro CRISPR-Cas9 system, reverse genetics systems of two mild coronaviruses HCoV-OC43 and HCoV-229E were rapidly established. Antiviral assays, high-content imaging, and NanoLuc luciferase assays were used to characterize reporter viruses and replicon systems. RESULTS: We rapidly assembled infectious clones for two mild coronaviruses, HCoV-OC43 and HCoV-229E, using transformation-associated recombination (TAR) cloning in yeast. The infected clones could stably express the mGreenLantern reporter gene. We further generated T7 promoter-driven RNA replicon of HCoV-229E and CMV promoter-driven DNA replicon of HCoV-OC43, with the readout of NanoLuc luciferase activity. The effectiveness of these tools for antiviral study was evaluated using the broad-spectrum RNA-dependent RNA polymerase inhibitor remdesivir, exhibiting high sensitivity, efficiency, and convenience. DISCUSSION: The application of yeast-based TAR cloning significantly facilitates the rapid assembly of large viral genome, and the establishment of HCoV-OC43 and HCoV-229E reverse genetics systems provides valuable platforms for studying the biology and developing antivirals against coronaviruses.