A yeast-based reverse genetics system to generate HCoV-OC43 reporter viruses encoding an eighth sgRNA

Coronaviruses have large, positive-sense single-stranded RNA genomes that challenge conventional strategies for mutagenesis. Here, we report the development of a new reverse genetics system for the endemic human coronavirus (HCoV) OC43 that utilizes transformation-associated recombination (TAR) to assemble complete viral genomes from dsDNA genome fragments via homologous recombination in Saccharomyces cerevisiae . Following cDNA synthesis from HCoV-OC43 viral RNA, we used TAR to capture fragments of the HCoV-OC43 genome to store as sequence-validated dsDNA parts. We performed combinatorial assembly in yeast to obtain an intact dsDNA copy of the HCoV-OC43 genome sufficient to launch viral replication upon introduction into human cells, yielding the yeast assembled OC43 ^YA virus. We also expanded the OC43 ^YA genome by inserting an eighth body transcription regulatory sequence (B-TRS) and an mClover3-H2B reporter gene between the M and N genes, designed to allow the reporter protein to be translated from its own subgenomic mRNA. We thoroughly evaluated OC43 ^YA and the OC43-mClo ^YA reporter virus, and demonstrated comparable viral gene expression, fitness in cell culture, and susceptibility to antivirals, compared to their natural progenitor. In summary, this new HCoV-OC43 reverse genetics system provides a modular platform for mutagenesis and combinatorial assembly of HCoV-OC43 genomes, and demonstrates the feasibility of expanding the genome while avoiding disruption of native coding sequences.