The oncoprotein Tax of the human T-cell leukemia virus type 1 activates transcription via interaction with cellular ATF-1/CREB factors in Saccharomyces cerevisiae.
The transcription factor Tax of the oncogenic human T-cell leukemia virus type 1 is likely to be responsible for viral replication in the host organism and for the induction of proliferation in infected cells. To investigate Tax-mediated transcription in vivo, we expressed Tax as well as CREB in Saccharomyces cerevisiae. The activity of these proteins was monitored by expression of a beta-galactosidase reporter gene, which was fused to two viral 21-bp repeats located upstream of the yeast cytochrome c1 oxidase minimal promoter. Coexpression of Tax and CREB in S. cerevisiae led to a 20-fold increase in beta-galactosidase activity in comparison with that in strains expressing either Tax or CREB alone. By screening a human cDNA library, we were able to demonstrate that the Tax transactivation assay using S. cerevisiae can be successfully applied to identify other cellular proteins forming ternary complexes with Tax and 21-bp repeats in vivo. Upon transformation in S. cerevisiae, 1 of 13,500 clones tested positive. Sequencing of the cDNA insert of the rescued plasmid revealed that this DNA encoded the ATF-1 protein. beta-Galactosidase induction was comparable to that of the Tax/CREB coexpression system. This indicates that Tax-mediated transcription is critically dependent on the presence of cellular CREB or ATF-1 in vivo. Stimulation of transcription initiation required an unmasked NH2 terminus of Tax. Fusion of Tax to the yeast Gal4 protein abolished the transactivation potential of Tax. Reconstitution of the transcriptional properties of viral Tax together with the cellular proteins of the ATF-1/CREB family in S. cerevisiae allows the functional characterization of these proteins in vivo.
Shnyreva
M
M
Department of Cell and Molecular Biology, Hans-Knoll-Institut für Naturstoff-Forschung e.V., Jena, Germany.
Munder
T
T
D051697:Activating Transcription Factor 1; D004268:DNA-Binding Proteins; D016356:Gene Products, tax / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000235:genetics
D051697:Activating Transcription Factor 1; D004268:DNA-Binding Proteins; D016356:Gene Products, tax; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D001616:beta-Galactosidase