Previously we reported that, unlike RNA of typical tobamoviruses, the translation of the coat protein (CP) gene of a crucifer-infecting tobamovirus (crTMV) in vitro occurred by an internal ribosome entry mechanism mediated by the 148-nt region that contained an internal ribosome entry site (IRESCP,148CR). The equivalent 148-nt sequence from TMV U1 RNA (U1(CP,148)(SP)) was incapable of promoting internal initiation. In the present work, we have found that the 228-nt region upstream of the movement protein (MP) gene of crTMV RNA (IRESMP,228CR) contained an IRES element that directed in vitro translation of the 3'-proximal reporter genes from chimeric dicistronic transcripts. Surprisingly, the equivalent 228-nt sequence upstream from the MP gene of TMV U1 directed translation of the downstream gene of a dicistronic transcripts as well. Consequently this sequence was termed IRESMP,228U1. It was shown that IRESMP,228CR, IRESMP,228U1, and IRESCP,148CR could mediate expression of the 3'-proximal GUS gene from dicistronic 35S promoter-based constructs in vivo in experiments on transfection of tobacco protoplasts and particle bombardment of Nicotiana benthamiana leaves. The results indicated that an IRES element was located within the 75-nt region upstream of MP gene (IRES,,,), which corresponded closely to the length of the 5'UTR of TMV subgenomic RNA (sgRNA) I-2. The RNA transcripts structurally equivalent to I-2 sgRNAs of TMV U1 and crTMV, but containing a hairpin structure (H) immediately upstream of IRESMP,75 (HIRESMP,75CR-MP-CP-3'UTR; HIRESMP,75U1-MP-CP-3'UTR), were able to express the MP gene in vitro. The capacity of HIRESMP,75CR sequence for mediating internal translation of the 3'-proximal GUS gene in vivo, in tobacco protoplasts, was demonstrated. We suggested that expression of the MP gene from I-2 sgRNAs might proceed via internal ribosome entry pathway mediated by IRESMP element contained in the 75-nt 5'UTR. Our results admit that a ribosome scanning mechanism of the MP gene expression from I-2 sgRNA operates concurrently. (C) 1999 Academic Press.
