FIG. 1.

Strategy for directionally assembling a TGEV infectious construct. (A) The TGEV genome is a linear positive-polarity RNA of about 28,500 nucleotides. Using RT-PCR and unique oligonucleotide primer mutagenesis, five clones spanning the entire TGEV genome were isolated using standard recombinant DNA techniques. Unique BglI sites were inserted at the junctions between each clone, a unique T7 start site was inserted at the 5′ end of clone A, and a 25-nucleotide T tail and downstream NotI site were inserted at the 3′ end of clone F. The approximate location of each site is shown. (B) Cloning the TGEV B amplicon. Because of chromosomal instability in E. coli, it was noted that two B clones (TGEV B1 and B3) contained large insertions at nucleotide 9973 in the TGEV genome (17). Other TGEV B clones had deletions across these sequences. Assuming that these insertions and deletions were “detoxifying” poison TGEV sequences in E. coli, we bisected the B fragment by inserting a BstXI site at position 9949 and cloning two separate clones designated TGEV B1-1,2 and TGEV B2-1,2. Quasispecies variation in the sequence of each independent plasmid clones is shown, with conserved changes denoted with asterisks. These conserved changes differed from the published sequence reported by Eleouet et al. (17) but were identical to the sequence reported by Almazan et al. (3). To reconstruct a wild-type B1 fragment, an SfiI-PflI fragment from B1-1 was isolated and inserted into the TGEV B1-2 backbone to produce a consensus B1 clone.