THE IE2 REGULATORY PROTEIN OF HUMAN CYTOMEGALOVIRUS INDUCES EXPRESSION OF THE HUMAN TRANSFORMING GROWTH-FACTOR-BETA-1 GENE THROUGH AN EGR-1BINDING-SITE

Increases in transforming growth factor beta 1 (TGF-beta 1) mRNA and b iological activity in the early phase of human cytomegalovirus (CMV) i nfection in fibroblasts are paralleled by increased TGF-beta 1-chloram phenicol acetyltransferase (CAT) reporter gene activity. To determine how CMV infection transactivates the TGF-beta 1 promoter, we examined the effects of the cotransfected IE2 regulatory protein of human CMV o n 5'-deleted TGF-P beta promoter-CAT reporter genes in transient DNA t ransfection assays. Two upstream TGF-beta 1 promoter regions each cont aining an Egr-1 consensus site were shown to he important for IE2-indu ced transactivation in a cell type that displayed greatly reduced nons pecific activity. Furthermore, transfer of an Egr-1 site from between positions -125 and -98, but not point mutant versions of this site, to a heterologous promoter also conveyed IE2 responsiveness. Addition of an IE2 expression vector or use of the U373 A45 astrocytoma cell line expressing IE2 also produced synergistic stimulation of GAL4-Egr-1-me diated activation of a target promoter containing GAL4 binding sites. The 80-kDa IE2 protein present in A45 cells proved to selectively bind to glutathione S-transferase (GST)-Egr-1 beads. The results of in vit ro protein binding assays also revealed that an intact in vitro-transl ated IE2 protein bound directly to the GST-Egr-1 fusion protein throug h the zinc finger domain of the Egr-1 protein and that this binding ac tivity was abolished by deletion of parts of the zinc finger DNA-bindi ng domain, Similarly, the Egr-1 protein was found to associate prefere ntially with a small region within the C-terminal half of the IE2 prot ein adjacent to the DNA-binding and dimerization domains that are impo rtant for both transactivation and downregulation. We conclude from th ese observations that IE2 may regulate transcription of the TGF-beta 1 gene as well as other potential cellular targets by virtue of its abi lity to interact with the Egr-1 DNA-binding protein.