Distinct viral sequence elements are necessary for expression of Tomato golden mosaic virus complementary sense transcripts that direct AL2 and AL3 gene expression.

Transient expression studies using Nicotiana benthamiana protoplasts and plants have identified sequences important for transcription of complementary sense RNAs derived from Tomato golden mosaic virus (TGMV) DNA component A that direct expression of AL2 and AL3. Transcription of two complementary sense RNAs, initiating at nucleotides 1,935 (AL1935) and 1,629 (AL1629), is directed by unique sequences located upstream of each transcription initiation site. One element is located between 28 and 124 nucleotides (nt) upstream of the AL1935 transcription start site, which differs from a second element located 150 nt downstream, between 129 and 184 nt upstream of the AL1629 transcription start site. Transcription initiation at nucleotide 1,935 is lower than that at nucleotide 1,629 as determined by run-on transcription assays, and the resulting transcript is only capable of expressing AL3. The transcript initiating at nucleotide 1,629 is capable of directing expression of both AL2 and AL3, although expression of AL3 is up to fourfold greater than that for AL2. Nuclear factors purified from tobacco suspension cells bind to sequences upstream of both AL1935 and AL1629, correlating with the ability of these sequences to direct gene expression. Thus, in tobacco, regulatory sequences direct transcription of two unique TGMV messenger RNAs that differentially express AL2 and AL3.