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 OC43YA virus. We also expanded the OC43YA 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 OC43YA and the OC43-mCloYA 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. IMPORTANCEHuman coronavirus OC43 (HCoV-OC43) is an endemic human coronavirus that typically causes relatively mild respiratory illnesses and displays seasonal patterns of infection. We developed a new system to assemble DNA copies of HCoV-OC43 genomes and generate recombinant viruses for research purposes. This system uses yeast, first to capture segments of DNA encompassing the entire RNA-based viral genome, and then to stitch them together into complete DNA genome copies that can be amplified in bacteria and introduced into human cells to initiate an infectious cycle, ultimately yielding recombinant viruses with comparable properties to their natural progenitors. We also devised a strategy to expand the viral genome, adding a gene for a reporter protein encoded by an additional eighth subgenomic mRNA. This yeast-based genome assembly system provides a modular platform for rapid mutagenesis and combinatorial assembly of HCoV-OC43 genomes and demonstrates the feasibility of expanding the genome.