The regulation of varicella-zoster virus (VZV) gene expression is largely controlled at the transcriptional level by a few key viral proteins cooperating with one another and with cellular transcription factors. However, the mechanisms involved are largely unclear. To identify the sequences important for the transcriptional regulation of open-reading frame (ORF) 4, itself encoding a transcriptional regulator, a mutation analysis of the promoter was done. These studies identified an element between −69 and −59 (relative to the transcriptional start site), which was critical to the activity of the promoter upon stimulation by the VZV transactivator IE62 and by VZV infection. DNA-protein interaction studies revealed that VZV induced the binding of a specific protein complex to this element, which contained the ubiquitous transcription factor USF. ORF 4 is the second VZV gene (in addition to VZV ORF 29) in which USF binding plays a critical role in gene expression.