FIG. 1.

Organization of the gene constructs used for the present study. (A) Plasmid pB2-14 contains the complete cDNA for BNYVV RNA-2 in pBluescribe (4, 53). Only ORF1 and ORF2 are represented. Amino acid (aa) numbers are indicated. (B) In plasmids pB2-RT-GFP1 and pB2-RT-GFP2 (15), the GFP gene replaces the AccI₁₄₁₅-AccI₁₈₂₈ sequence of pB2-14, truncating the TM2 domain; in pB2-RT-GFP3, the GFP gene is inserted in frame into the AccI₁₄₁₅ site of pB2-14. (C) pB2-RT-Δ50-GFP2 derives from pB2-RT-GFP2 and carries a deletion of nucleotides 793 to 894, truncating the MTS domain. (D) The MTS, IS, TM1, and TM3-TM4 domains, individually or in combination, were inserted in frame with the GFP gene between the upstream and downstream noncoding sequences of the cDNA for BNYVV RNA-3 in the pRep replicon (16). (E) The GFP gene alone or the MTS-IS-TM1 or IS-TM1 domain fused to the GFP gene was inserted into the expression cassette of the pΩ plasmid (41). (F) In pB2-UAU, the CP sequence ends with a tyrosine codon, allowing full readthrough, whereas in pB2-TS the CP sequence ends with three stop codons, preventing readthrough; pB2-UAU-ΔTM1 carries a deletion of nucleotides 961 to 1053, eliminating the TM1 domain. *, stop codons; “(CC)” indicates that two extra C residues were added at the end of the GFP sequence to ensure the in-frame insertion; UAU, the stop codon of ORF1 was mutated to a UAU tyrosine codon. For more details, see Materials and Methods.