Increases in transforming growth factor β1 (TGF-β1) mRNA and biological activity in the early phase of human cytomegalovirus (CMV) infection in fibroblasts are paralleled by increased TGF-β1-chloramphenicol acetyltransferase (CAT) reporter gene activity. To determine how CMV infection transactivates the TGF-β1 promoter, we examined the effects of the cotransfected IE2 regulatory protein of human CMV on 5'-deleted TGF-β1 promoter-CAT reporter genes in transient DNA transfection assays. Two upstream TGF-β1 promoter regions each containing an Egr-1 consensus site were shown to be important for IE2-induced transactivation in a cell type that displayed greatly reduced nonspecific 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-mediated 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 heads. The results of in vitro protein binding assays also revealed that an intact in vitro-translated IE2 protein bound directly to the GST-Egr-1 fusion protein through the zinc finger domain of the Egr-1 protein and that this binding activity was abolished by deletion of parts of the zinc finger DNA-binding domain. Similarly, the Egr-1 protein was found to associate preferentially with a small region within the C-terminal half of the IE2 protein adjacent to the DNA-binding and dimerization domains that are important for both transactivation and downregulation. We conclude from these observations that IE2 may regulate transcription of the TGF-β1 gene as well as other potential cellular targets by virtue of its ability to interact with the Egr-1 DNA- binding protein.
ASJC Scopus subject areas
- Insect Science