Like other Alphaherpesvirinae subfamily members, bovine herpesvirus 1 (BHV-1) establishes latency in sensory neurons. The latency-related RNA (LR-RNA) is abundantly expressed in latently infected sensory neurons. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) does not reactivate from latency, in part because it induces higher levels of apoptosis in infected neurons. ORF2 is not the only viral product expressed during latency, but it is important for the latency reactivation cycle because it inhibits apoptosis. In this study, a yeast 2-hybrid screen revealed that ORF2 interacted with two cellular transcription factors, Notch1 and Notch3. These interactions were confirmed in mouse neuroblastoma cells by confocal microscopy and in an in vitro "pulldown" assay. During reactivation from latency, Notch3 RNA levels in trigeminal ganglia were higher than those during latency, suggesting that Notch family members promote reactivation from latency or that reactivation promotes Notch expression. A plasmid expressing the Notch1 intercellular domain (ICD) stimulated productive infection and promoters that encode the viral transcription factor bICP0. The Notch3 ICD did not stimulate productive infection as efficiently as the Notch1 ICD and had no effect on bICP0 promoter activity. Plasmids expressing the Notch1 ICD or the Notch3 ICD trans-activated a late promoter encoding glycoprotein C. ORF2 reduced the trans-activation potential of Notch1 and Notch3, suggesting that ORF2 interfered with the trans-activation potential of Notch. These studies provide evidence that ORF2, in addition to inhibiting apoptosis, has the potential to promote establishment and maintenance of latency by sequestering cellular transcription factors.