Defective interfering particles (DIP) of equine herpesvirus 1 (EHV-1) inhibit standard virus replication and mediate persistent infection. The DIP genome is comprised of only three genes: UL3, UL4, and a hybrid gene composed of portions of the IR4 (EICP22) and UL5 (EICP27) genes. The hybrid gene is important for DIP interference, but the function(s) of the UL3 and UL4 genes are unknown. Here, we show that UL4 is an early gene activated solely by the immediate early protein. The UL4 protein (UL4P) was detected at 4hours post-infection, was localized throughout the nucleus and cytoplasm, and was not present in purified virions. EHV-1 lacking UL4P expression was infectious and displayed cell tropism and pathogenic properties in the mouse model similar to those of parental and revertant viruses. Reporter assays demonstrated that the UL4P has a broad inhibitory function, suggesting a potential role in establishing and/or maintaining DIP-mediated persistent infection.