Tetracycline-mediated regulation of gene expression within the human cytomegalovirus genome

To evaluate the utility of tetracycline gene regulation in the study of human cytomegalovirus gene functions, expression of luciferase under the control of tetracycline-regulatable promoters was studied following transient plasmid transfections and from within recombinant human cytomegalovirus genomes. The tetracycline-regulatable promoter PhCMV*-1 contains sequences from the human cytomegalovirus ie1/ie2 promoter and seven upstream tet operator sires which bind the activator protein tTA only in the absence of tetracycline (Gossen and Bujard (1992). Proc. Natl. Acad. Sci. USA 89, 5547-5551). Two modifications of PhCMV*-1 were also studied: P-1129, in which the tet operator sites were reduced from seven to one; and P-1125, in which human cytomegalovirus sequences were replaced by adenovirus major late promoter and terminal deoxynucleotidyltransferase initiator sequences. In transient assays, PhCMV*-1 and P-1125 exhibited modest differential regulation but were strongly activated by viral infection. P-1129 exhibited less viral activation and narrower regulation. In the viral genome, PhCMV*-1 exhibited regulation up to 7-fold during late limes of infection, whereas P-1125 displayed nearly 100-fold regulation. Regulation of P-1125 was fully reversed within 12 to 24 h of adding or removing tetracycline. These results suggest that P-1125 may provide sufficient conditional expression to effectively regulate human cytomegalovirus late genes, (C) 1999 Academic Press.