Figure 3
In vivo translation assays.
(a) Reporter constructs with various terminal nt sequences flanking the firefly luciferase gene (F-Luc). LUC-TMV: 5′ and 3′ NCRs of TMV30BGFP49 RNA; LUC-R1: 5′ and 3′ NCRs of LCV RNA1; LUC-R2A: 5′ and 3′ NCRs of LCV RNA2; LUC-R2A(−), LUC-R2B and LUC-R2C: essentially LUC-R2A except that in LUC-R2A(−), the 3′ NCR is replaced by 98 nts from the GFP gene (striped box) and in LUC-R2B and LUC-2C, the 3′ NCR is extended by adding 9 and 302 nts, respectively, from the immediate upstream region of the LCV RNA 2 3′ NCR; LUC-R2CΔSL1 and LUC-R2CΔSL2: essentially LUC-R2C with stem-loop (SL)1 and SL2, respectively, of the Y-shape structure (YSS) deleted; and LUC-R2D and LUC-R2E: essentially LUC-R2A and LUC-R2C, respectively, except that the 5′ NCR is extended by adding 99 nts from the proximal 5′ end of the P5.6 ORF of LCV RNA2. Complete YSS (black bar labeled “YSS”), partial YSS (unlabeled black bar) and YSS with deleted SL1 (ΔSL1) or SL2 (ΔSL2) are indicated. Light gray and dark gray boxes represent non-coding and coding sequences, respectively. Numbers above the vertical lines in the constructs, except for those at the 3′ flanking region of LUC-R2A(−), are the nt positions in the genomic RNAs of the respective viruses. ((b,c) [bottom]) In vivo translation assays. Protoplast inoculations were performed using the in vitro transcripts of Renilla luciferase (R-Luc) and those of F-Luc reporters or water (w; mock inoculation) as indicated; in ((c) [bottom]), protoplasts were co-inoculated with the in vitro transcripts of cloned LCV RNA1. Means (means <0.009 are not indicated) and standard errors are from relative F-Luc/R-Luc activities determined from triplicate experiments. ((c) [top]) Northern analysis of total RNA extracted from protoplasts subjected to the treatments in ((c) [bottom]). The polarity of F-Luc RNA being probed is as indicated. Insets (C1 and C2) are 24 hr exposures for each blot. Sizes of RNAs were estimated based on methylene blue-stained RNA standards shown on the left of each blot. The methylene blue-stained 25S rRNA of each sample was included to demonstrate the equal loading of total RNA samples.
