a, General workflow of TAR cloning and virus rescue. In-yeast genome reconstruction requires one-step delivery of overlapping DNA fragments that cover the viral genome and a TAR vector in yeast. Viral ORFs and the ORF for GFP are indicated. Transformed DNA fragments are assembled by homologous recombination in yeast to generate a YAC that contains the full-length viral cDNA sequence. In vitro production of infectious capped viral RNA starts with the isolation of the YAC, followed by plasmid linearization to provide a DNA template for run-off T7 RNA polymerase-based transcription. Virus rescue is initiated by electroporation of BHK-MHV-N cells, after which virus production and amplification is carried out by culturing the virus with susceptible cells. b, Recovery of infectious rMHV-GFP from yeast clones 1 and 2. Cell-culture supernatants—which contain viruses produced after virus rescue of two MHV-GFP YAC clones—were used to infect 17Cl-1 cells. At 48 h after infection, infected cells were visualized for GFP expression (top) and by bright-field microscopy (bottom). Mock represents 17Cl-1 cells inoculated with the supernatant from BHK-MHV-N cells electroporated without viral RNAs. Images are representative of two independent experiments. Scale bars, 100 μm. c, Replication kinetics of parental MHV-GFP and rMHV-GFP clones 1 and 2. L929 cells were infected (multiplicity of infection (MOI) = 0.1), and cell-culture supernatants were collected at the indicated time points after infection and titrated by plaque assay. PFU, plaque forming units. Data represent the mean ± s.d. of three independent biological experiments (n = 3). Statistical significance was determined by two-sided unpaired Student’s t-test without adjustments for multiple comparisons. NS, not significant. P values (from left to right): top, NS, P = 0.2905; NS, P = 0.3504; NS, P = 0.1817; NS, P = 0.9862; NS, P = 0.6738; bottom, NS, P = 0.0835; NS, P = 0.1400; NS, P = 0.2206; NS, P = 0.8020; NS, P = 0.5894.