A TEF-1-element is required for activation of the promoter of pseudorabies virus glycoprotein X gene by IE180.

The pseudorabies virus (PRV) immediate-early regulatory protein IE180 is able to transactivate the viral early and late genes. Using chloramphenicol acetyltransferase (CAT) assay, we investigated the transactivation function of IE180 to the promoter of PRV glycoprotein X (gX) gene, and our results showed that IE180 could significantly increase the expression of CAT gene which was under the control of gX promoter. To further identify the activation domains of IE180 protein that interact with the gX promoter sequences, various truncated mutants of IE180 gene and gX promoter gene were constructed and analyzed by CAT and gel retardation assay. Results revealed that the N-terminal amino acid residues from 133 to 736 of IE180 could interact with the binding site of transcriptional enhancer factor-1 (TEF-1) that resides in the gX promoter. Formation of protein-DNA complexes between the IE180 protein and the TEF-1 element of the gX promoter was observed using electrophoretic mobility shift assay (EMSA) as well as Southwestern blot analysis. These results indicated that a direct interaction occurred between IE180 and the TEF-1 element; and this interaction was abolished if the TEF-1 element was mutated. The association of IE180 with the TEF-1 element was further confirmed by the supershift of EMSA complexes using IE180 specific antibody. Taken together, our results suggested that formation of a complex between the IE180 protein and TEF-1 element in the gX promoter region was involved in the transcriptional regulation of the gX gene.