HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36

Human cytomegalovirus (HCMV) establishes latency in CD34⁺ hematopoietic progenitor cells (HPCs), where reactivation is intimately linked to cellular differentiation. We demonstrate that the Notch signaling pathway, a key regulator of stem cell maintenance and differentiation, functions as a barrier to HCMV reactivation. Two viral gene products, UL8 and miR-UL36, modulate this pathway during reactivation. UL8 promotes degradation of the Notch3 receptor via the endosomal/lysosomal pathway, dependent on two tyrosine-based motifs (Y305/314) in its cytoplasmic tail. A UL8 mutant lacking these motifs fails to degrade Notch3, resulting in sustained Notch signaling and impaired reactivation <i>in vitro</i> and in humanized mice. Similarly, miR-UL36 reduces the expression of Notch3 and the Notch transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBPJ), suppressing Notch signaling. Deletion of miR-UL36 inhibits reactivation, but this defect, like that of the UL8 mutant, can be rescued by pharmacologic Notch inhibition. Thus, HCMV employs multiple gene products to suppress Notch signaling and promote conditions conducive to reactivation. These findings reveal how HCMV manipulates host differentiation pathways to control latency and suggest therapeutic strategies to prevent viral recurrence in immunocompromised patients.IMPORTANCEHuman cytomegalovirus (HCMV) establishes lifelong latency, posing significant risks to transplant recipients and other immunocompromised individuals. Reactivation depends on progenitor cell differentiation; however, the viral mechanisms governing this process remain unclear. We identify Notch signaling as a major inhibitory pathway to reactivation and show that HCMV uses UL8 and miR-UL36 to suppress this pathway. UL8 degrades Notch3, while miR-UL36 downregulates Notch3 and RBPJ, together reducing Notch signaling and enabling reactivation. Mutant viruses lacking these regulators fail to reactivate efficiently, but this can be reversed by pharmacological inhibition of Notch. These findings establish Notch pathway suppression as a critical viral strategy for reactivation and highlight potential therapeutic targets for preventing HCMV disease.