Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors

A series of tobacco mosaic virus (TMV)-based hybrid vectors for transient g ene expression were constructed with similar designs but differing in the s ource of heterologous tobamovirus sequence: Odontoglassum ringspot virus, t obacco mild green mosaic virus variants U2 and U5, tomato mosaic virus, and sunn-hemp mosaic virus. These vectors contained a heterologous coat protei n subgenomic mRNA promoter and coat protein open reading frame (ORF) and ei ther TMV or heterologous 3' nontranslated region. The foreign ORF, from the jellyfish green fluorescent protein (GFP) gene, was transcribed from the n ative TMV coat protein subgenomic mRNA promoter, which extended into the co at protein ORF. The presence of an in-frame stop codon within the GFP mRNA leader and the choice of sequence of GFP ORFs substantially affected transl ational efficiency. However, the major regulatory component of gene express ion in these Vectors appeared to be transcriptional rather than translation al. There was an inverse relationship between expression of GFP and the het erologous coat protein genes that was reflected in accumulation of the resp ective mRNAs and proteins. The most effective vector in this series (30B) c ontained sequences encoding the coat protein subgenomic mRNA promoter, coat protein ORF, and 3' nontranslated region from tobacco mild green mosaic vi rus U5. Expressed from 30B, GFP accumulated up to 10% of total soluble prot ein in leaves. (C) 1999 Academic Press.