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

Area of Science:

• Virology
• Molecular Biology
• Yeast Genetics

Background:

• Coronaviruses possess large RNA genomes challenging conventional mutagenesis.
• Human coronavirus OC43 (HCoV-OC43) is a model betacoronavirus causing mild respiratory illness.
• Transformation-associated recombination (TAR) in yeast is effective for manipulating large viral genomes.

Purpose of the Study:

• To develop an updated transformation-associated recombination (TAR) assembly and mutagenesis system for human coronavirus OC43 (HCoV-OC43).
• To enable rapid generation and modification of HCoV-OC43, including reporter viruses.
• To facilitate studies on betacoronavirus biology and host-pathogen interactions.

Main Methods:

• Synthesized cDNA from HCoV-OC43 RNA.
• Generated five plasmids containing ~7.2 kb portions of the viral genome.
• Assembled complete viral genomes using homologous recombination in *Saccharomyces cerevisiae* (yeast).
• Rescued wild-type and fluorescent reporter viruses (OC43-mClover3-H2B, OC43-mRuby3-H2B, OC43-mCardinal).

Main Results:

• Successfully assembled and rescued full-length HCoV-OC43 plasmids.
• Generated functional wild-type and reporter viruses, including OC43-mClover3-H2B.
• OC43-mClover3-H2B replicated comparably to reference strains.
• Reporter virus produced protein from a novel subgenomic RNA without deleting viral genes.

Conclusions:

• The updated HCoV-OC43 reverse genetics system enhances the study of betacoronavirus biology.
• This system facilitates the generation of reporter viruses for monitoring viral replication.
• It accelerates research into host-pathogen interactions and the development of novel antivirals.