FIG. 6.

Northern blot analysis of recombinant TGEV viruses carrying the GFP gene under different TRSs and at different positions of the viral genome. (A) Schematic structure of the cDNAs encoding the rTGEV-Δ3-TRSn-GFP-7 and rTGEV-Δ3-N-TRSn-GFP-7 RNAs with the GFP gene under the 5′ TRS of the N gene inserted at place where ORFs 3a and 3b were deleted or between genes N and 7, respectively. The numbers and letters inside the rectangles indicate the viral genes. CMV, cytomegalovirus immediate-early promoter; An, poly(A); HDV, hepatitis delta virus ribozyme; BGH, bovine growth hormone termination and polyadenylation signals. (B) Northern blot analysis of intracellular RNAs extracted at passage 2 from cells infected with the wild-type TGEV virus (lane 1), rTGEV-Δ3 (lane 2), rTGEV-Δ3-TRS3a-GFP (lane 3), rTGEV-Δ3-TRSn-GFP-7 (lane 4), rTGEV-Δ3-N-TRSn-GFP-7 clone 3 (lane 5), or rTGEV-Δ3-N-TRSn-GFP-7 clone 12 (lane 6). Hybridization was performed with a probe complementary to the 3′ untranslated region of the genome. Numbers and letters on the left indicate the position of the genomic (gRNA) and viral mRNAs. S, spike; 3a, gene 3a; E, envelope; M, membrane; N, nucleoprotein; 7, gene 7. The positions of the new sgmRNAs generated from recombinant viruses are indicated by arrows on the right side of the figure. sgmRNA-GFP, sgmRNA encoding GFP gene; sgmRNA-Δ3, sgmRNA synthesized from the TRS of ORF 3a in the deletion mutant rTGEV-Δ3; sgmRNA-Δ7, sgmRNA synthesized by rTGEV-Δ3-N-TRSn-GFP-7 clone 3 from a noncanonical junction site. Open arrowheads and symbols indicate sgmRNAs of rTGEV-Δ3-N-TRSn-GFP-7 clone 3 with detectable mobility differences relative to the corresponding sgmRNAs of wild-type TGEV or rTGEV-Δ3 (solid arrowheads and symbols).