Assembly and Mutagenesis of Human Coronavirus OC43 Genomes in Yeast via Transformation-Associated Recombination.

Abstract

Human coronavirus OC43 (HCoV-OC43) is an endemic "common cold" coronavirus widely used to study fundamental aspects of coronavirus biology and to test therapeutic interventions. Recently, we used a yeast-based reverse genetics strategy to create recombinant HCoV-OC43 and fluorescent reporter viruses. We assembled a DNA copy of the HCoV-OC43 genome from six linear dsDNA fragments and a linearized yeast centromeric plasmid/bacterial artificial chromosome (YCpBAC) vector in Saccharomyces cerevisiae using transformation-associated recombination (TAR). Reporter genes encoding mCardinal fluorescent protein or histone H2B fused to mClover3 (mClover-H2B) or mRuby3 (mRuby-H2B) were inserted into an intergenic region between the HCoV-OC43 M and N genes. Assembled full-length HCoV-OC43-encoding plasmids were delivered into permissive mammalian cells to initiate viral gene expression, genome replication, and production of infectious progeny. This technique allows for the precise mutagenesis of any area of the HCoV-OC43 genome using homologous recombination, yielding genetically defined reference plasmids for the future generation of HCoV-OC43 virus stocks. Key features • Utilizes the previously developed TAR assembly method [1] to assemble and mutagenize a double-stranded DNA copy of the single-stranded RNA HCoV-OC43 genome [2]. • The availability of multiple sub-genomic and full-length HCoV-OC43-encoding plasmids provides flexibility in how substitution or deletion mutations can be incorporated using PCR or restriction cloning. • Reporter viruses enable rapid visualization and quantification of infection. • Generate and isolate a mutagenized HCoV-OC43 plasmid in approximately 14 days, followed by rescue of infectious virus in an additional 12-16 days.