A yeast-assembled, plasmid-launched reverse genetics system for the murine coronavirus MHV-A59

The Betacoronavirus murine hepatitis virus (MHV) is an important model system for studying coronavirus (CoV) molecular and cell biology. Despite this, few reagents for MHV are available through repositories such as ATCC or Addgene, potentially limiting the widespread adoption of MHV as a tractable model system. To overcome some challenges inherent in the existing MHV reverse genetics systems, we developed a plasmid-launched transformation-associated recombination (TAR) cloning-based system to assemble the MHV (strain A59; MHV-A59) genome. Following assembly in yeast, virus replication was launched by transfecting the fully assembled genome into HEK-293T cells. MHV-A59 recovered using this TAR cloning-based approach (WT_TAR MHV-A59) replicated with kinetics identical to the virus recovered using a ligation- and T7-based approach (WT_LIG MHV-A59). Additionally, WT_TAR MHV-A59 can be detected at least 10 h post-transfection without requiring additional nucleocapsid (N) provided in trans. Lastly, we demonstrated the tractability of this TAR cloning-based system by recovering MHV-A59 expressing an 11 amino acid-containing HiBiT tag fused to the C-terminus of spike (S). While this virus, S_C MHV-A59, replicated with reduced kinetics compared to WT_TAR MHV-A59, the kinetics of virion production could be measured over time directly from the supernatant. This report represents the first plasmid-launched, TAR cloning-based system for MHV-A59. Furthermore, it describes a new reporter virus that could be used to study early steps during MHV-A59 entry and be used in the screening of antiviral compounds. To support future research with MHV-A59, we have made the necessary plasmids for this system available through ATCC.