FIG. 1.

Selection of the MΔ2 mutant. (A) Construction of a transcription vector for synthesis of donor RNA. Plasmid pLK61 was derived from pMH54 (18) as detailed in Materials and Methods. The restriction sites shown are those relevant to plasmid construction (XhoI, EcoRV, BssHII, NheI, and BclI), in vitro transcription (PacI), or mutant analysis (AccI). The filled circle denotes the linker between the cDNA segments corresponding to the 5′ and 3′ ends of the MHV genome, and the arrow indicates the T7 promoter. The expanded region of sequence shows the three base changes made in pLK61 to create a stop codon at codon 227 of the M ORF and a new AccI site (underlined). The corresponding wild-type (wt) sequence from pMH54 is aligned for comparison. Also indicated is TRS7, the TRS governing synthesis of sgRNA7 (N mRNA). (B) Scheme for generation of the MΔ2 mutant by targeted RNA recombination between the interspecies chimera fMHV (18) and donor RNA transcribed from plasmid pLK61. fMHV contains the ectodomain-encoding region of the FIPV S gene (shaded rectangle) and is able to grow in feline cells but not in murine cells. A single crossover (solid line), within the HE gene, should generate a recombinant that has simultaneously reacquired the MHV S ectodomain and the ability to grow in murine cells and has also incorporated the mutant M gene (hatched rectangle). A potential second crossover (broken line), in gene 4 or the E gene, would exclude the mutant M gene from the recombinant. At the bottom is shown the mixed progeny of two independent targeted recombination experiments, forming tiny and large plaques on mouse L2 cells at 72 h postinfection.