The latency-associated transcript (LAT) gene is the only viral genomic region that is abundantly transcribed during pseudorabies virus (PrV) latent infection. The mechanism of reactivation of PrV from latency remains unknown. To analyze the regulation mechanism of the LAT promoter, we constructed a series of recombinant vectors in which various sequences upstream of LAT were linked to the chloramphenicol acetyltransferase (CAT) gene. Transcriptional efficiency was examined by cotransfection with plasmids carrying the PrV IE, EP0, or gD gene, respectively. Results showed that the activity of PrV LAT promoter was dramatically repressed by the IE180 protein and a TATA box and a putative IE180 binding site within the promoter were involved in this repression. To dissect the functional domains of IE180, we compared the relative repressive abilities of IE180 variants to the LAT promoter by transient transfection assays. Mutational analysis demonstrated that almost the whole IE180 (amino acid residues 1-1440) are essential for its repression to LAT promoter. To explore the possible mechanism of repression, an electrophoretic mobility shift assay (EMSA) using nuclear extracts from neuronal cells was performed and formation of protein-DNA complexes between IE180 and the oligonucleotide probe (-46 to -19, relative to the start site of LAT transcription) was demonstrated. The association of IE180 with the region encompassing the putative IE180 binding site and the TATA box upstream of PrV LAT gene was further confirmed by supershift of EMSA complexes using IE180 specific antibody. Thus, our results suggested that IE180 repressed the LAT promoter via an interaction between IE180, LAT promoter and cellular protein(s).