FIG 7.

Long-range base pairing between the DRTE and 5′ end of C-rich domain. (A) Alignment of potentially complementary regions of the C-rich motif adjacent to the leaky stop codon (lightface) and the portion of the DRTE adjacent to the conserved EXG[X]₆DE motif. The bases coding for DE (GAYGAR) are in lightface, with exceptions in lowercase. Complementary bases are in underlined magenta, base positions are indicated at ends, and the gap length between the two regions is in parentheses. Regions in the PLRV sequence highlighted in yellow indicate the bases deleted in the Δ4222-4236 and Δ4897-4911 mutants. Abbreviations not defined elsewhere: TobV2, Tobacco virus 2; CRLV, Carrot red leaf virus; TVDV, Tobacco vein distortion virus; BMYV, Beet mild yellowing virus; TuYV, Turnip yellows virus; BCV, Beet chlorosis virus; BrYV, Brassica yellows virus; WYDV, Wheat yellow dwarf virus; MABYV, Melon aphid-borne yellows virus; CLRDV, Cotton leafroll dwarf virus; CABYV, Cucurbit aphid-borne yellows virus; PeVYV, Pepper vein yellows virus; CpCSV, Chickpea chlorotic stunt virus; LABYV, Luffa aphid-borne yellows virus; PABYV, Pepo aphid-borne yellows virus; SCYLV, Sugarcane yellow leaf virus; MYDV-RMV, Maize yellow dwarf virus-RMV; WLYaV, Wheat leaf yellowing-associated virus; WCMV, White clover mottle virus; CpPV, Chickpea polerovirus; SPV1, Strawberry polerovirus 1; IxYMV1, Ixeridium yellow mottle virus 1; AEYV, African eggplant yellows virus; SYV, Sauropus yellowing virus; BLRV, Bean leafroll virus; SbDV, Soybean dwarf virus; ChaLV, Cherry-associated luteovirus; PaLV, Peach-associated luteovirus; AaLV, Apple-associated luteovirus; NSPaV, Nectarine stem-pitting-associated virus; RSDaV, Rose spring dwarf-associated virus; PEMV1, Pea enation mosaic virus 1; AEV1, Alfalfa enamovirus 1; CVEV, Citrus vein enation virus 1; GEV1, Grapevine enamovirus 1. (B) Diagram of the wild-type PLRV and mutants used for testing the effect of the complementary long-range base-pairing. Mut4222–4236-AA, replacement of nine out of 15 nucleotides in the 4222–4236 domain and no changes of original amino acids. Mut4222–4236-AA-C, complementary mutant of Mut4222–4236-AA in the DRTE; Mut4233–4236-AGGA: replacement of GCCU by AGGA in the domain of 4233–4236. Mut4233–4236-AGGA-C, complementary mutant of Mut4233–4236-AGGA. Arrows indicated the amino acid changes. (C) Western blot analysis of proteins in agroinfiltrated N. benthamiana tissue. Δ4222–4236, deletion of nt 4222 to 4236 in PLRV C-rich domain; Δ4897–4911, deletion of nt 4897 to 4911 in the DRTE; Δ4222–4233, deletion of nt 4222 to 4233. All the deletion mutations also did not change the ORF. Relative readthrough (Rel. RTP/CP) was calculated as the RTP/CP ratio, with that for WT PLRV set as 100%. Values represent the means (± standard error) determined from three independent experiments.