FIG. 1.

TGEV recombinant viruses expressing GFP. A series of TGEV recombinant viruses was generated using the TGEV-GFP2PflMI construct as the backbone for further characterization of the 3a and N gene TRS. (A) The TGEV-GFP2PflMI parent construct contains an ORF 3a deletion and insertion of GFP under the control of the 3a TRS and 20 nt of the N gene TRS. The location of the N and/or ORF 3a TRS sequences upstream of gfp is indicated. Clones were identified by DNA sequencing using an ABI model 377 automated sequencer, and the resulting constructs were subsequently used in the assembly of recombinant TGEV viral cDNAs. (B) TGEVmut1 was constructed by deleting nt 24709 to 24804 of the TGEV genome from TGEV-GFP2PflMI, corresponding to the ORF 3a CS and 89 nt of its 5′ flanking sequence. (C) TGEVmut2 contains the mutation of the upstream ORF 3a CS (ACUAAAC→GCUACAC), such that only the N CS is positioned upstream of gfp. (D) TGEVmut3 contains the 5′ N CS flanking sequence, which has been extended from 8 to 47 nt, (corresponding to nt 26858 to 26904 of the TGEV genome; GenBank accession no. AJ271965) while leaving the upstream ORF 3a CS completely intact. (E) TGEVmut4 contains the insertion of a nonspecific 39-nt sequence just 5′ of the 20 nt N gene TRS. (F) TGEVmut5 was engineered to contain unique StuI and Esp3I restriction sites which flank the CS element upstream of the N gene, and unique BstEII and PacI sites were introduced at the 3′ end of the N gene. The PacI restric-tion site was introduced at nucleotide position 28063 of the TGEV genome by mutagenesis of the Hp gene CS (ACTAAAC→ATTAATTAA). The wild-type Hp CS and gene were then repositioned just downstream of this PacI site, including 10 bp of upstream 5′ flanking sequence (nt 28051 to 28060). (G) To generate TGEVmut6, a ∼500-bp amplicon representing the CS deletion and 5′-truncated Hp gene was generated using the PacI site containing the 5′ primer (5′-TTA ATT AAA CCG GTT CGT CTT CCT CCA TGC TG-3′) and a 3′ primer within the cloning vector (Topo XL TA cloning vector; Invitrogen. Using the unique PacI site, this amplicon was inserted into the TGEVmut5 background to replace the wild-type Hp gene, resulting in a new recombinant TGEV F fragment containing a deletion of the CUAAAC Hp CS and 7 nt of 3′ flanking sequence (nt 28051 to 28074), including the first 4 nt of the Hp ORF. The only two available ATG start codons are out of frame with respect to the Hp ORF at nt positions 28087 and 28186 and would encode a seven- and nine-amino-acid protein, respectively.