Stabilization of the human cytomegalovirus UL136p33 reactivation determinant overcomes the requirement for UL135 for replication in hematopoietic cells.

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Human cytomegalovirus (HCMV) is a beta herpesvirus that persists indefinitely in the human host through a latent infection. The polycistronic UL133-UL138 gene locus of HCMV encodes genes regulating latency and reactivation. While UL138 is pro-latency, restricting virus replication in CD34+ hematopoietic progenitor cells (HPCs), UL135 overcomes this restriction and is required for reactivation. By contrast, UL136 is expressed with later kinetics and encodes multiple proteins with differential roles in latency and reactivation. Like UL135, the largest UL136 isoform, UL136p33, is required for reactivation from latency in HPCs; viruses failing to express either protein are unresponsive to reactivation stimuli. Furthermore, UL136p33 is unstable, and its instability is important for the establishment of latency, and sufficient accumulation of UL136p33 is a checkpoint for reactivation. We hypothesized that stabilizing UL136p33 might overcome the requirement of UL135 for replication. We generated recombinant viruses lacking UL135 that expressed a stabilized variant of UL136p33. Stabilizing UL136p33 did not impact the replication of the UL135 mutant virus in fibroblasts. However, in the context of infection in HPCs, stabilization of UL136p33 strikingly compensated for the loss of UL135, resulting in increased replication in CD34+ HPCs and in humanized NOD-scid IL2Rγcnull (huNSG) mice. This finding suggests that while UL135 is essential for replication in HPCs, it functions largely at steps preceding the accumulation of UL136p33, and that stabilized expression of UL136p33 largely overcomes the requirement for UL135. Taken together, our genetic evidence indicates an epistatic relationship between UL136p33 and UL135, whereby UL135 may initiate events early in reactivation that drive the accumulation of UL136p33 to a threshold required for productive reactivation. IMPORTANCE Human cytomegalovirus (HCMV) is one of nine human herpesviruses and a significant human pathogen. While HCMV establishes a lifelong latent infection that is typically asymptomatic in healthy individuals, its reactivation from latency can have devastating consequences in the immunocompromised. Defining viral genes important in the establishment of or reactivation from latency is important to defining the molecular basis of latent and replicative states and in controlling infection and CMV disease. Here we define a genetic relationship between two viral genes in controlling virus reactivation from latency using primary human hematopoietic progenitor cells and humanized mouse models.