Fig. 1.

The 5′ UTR of TCV gRNA contains a pyrimidine-rich element that can promote translation of an internal ORF. (A) Genomic organization of TCV gRNA and two sgRNAs. p28 and p88, virus-encoded replicase; p8 and p9, movement proteins; CP, coat protein. The sequence of the 5′ UTR is shown. The initial location of the 5′ TE (positions 36 to 63) is indicated along with location of a mutation (36-63m0) used to determine the importance of the sequence. (B) Diagram of the single- and dual-luciferase reporter constructs. NruI and SspI restriction sites were used to linearize constructs for in vitro transcription to produce RNA fragments containing or lacking 3′ UTR sequences. Rluc, Renilla luciferase; Fluc, firefly luciferase; Amb, amber stop codon. (C) Assessment of the translation activity of the 5′ UTR and 5′ UTR fragments in RRL using the dual-luciferase constructs containing viral 5′ sequences in the absence (N) or presence (3′ UTR+) of viral 3′ sequences. Data from at least three replicate experiments are shown. Standard deviation bars are given. (D) Translational activity of the 36-63 region containing mutations within the polypyrimidine-rich region shown in panel A. 36-63R, random 28-nt sequence.