Internal Initiation of Translation Directed by the 5′-Untranslated Region of the Tobamovirus Subgenomic RNA I2

Abstract

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 (U1CP,148SP) 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 (IRESMP,75), which corresponded closely to the length of the 5′UTR of TMV subgenomic RNA (sgRNA) I2. The RNA transcripts structurally equivalent to I2 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 I2 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 I2 sgRNA operates concurrently.