Mechanisms of Human Cytomegalovirus Latency in Primary Human Hematopoietic Cells

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PROJECT SUMMARY Human cytomegalovirus (HCMV) is a β-herpesvirus that establishes a lifelong latent infection in its host. Reactivation from latency can result in life-threatening disease in immune deficient individuals, particularly stem cell and solid organ transplant recipients. Our research program has a long-standing focus on defining HCMV determinants and host interactions at the mechanistic core of latency and reactivation. In previous funding periods, we defined a polycistronic locus within the ULb' region of the HCMV genome that spans the genes UL133-UL138. Within this region, we have identified virus-coded repressors and activators of virus replication. UL138 represses replication for latency, whereas UL135 activates replication for reactivation, in part, by overcoming the repressive action of UL138. UL135 and UL138 regulate EGFR with opposing effects and together comprise a switch to control viral latency and reactivation by regulating host signaling. Recently, UL136 has emerged as a key modulator of this switch. UL136 encodes 5 alternant protein forms or isoforms (p33, p26, p25, p23, and p19, based on their molecular mass) that differ only in their N-terminal sequences. UL136 isoforms accumulate at later times relative to UL135 and UL138 in productive infection, requiring the onset of HCMV genome synthesis for maximum expression. Some UL136 isoforms are required for reactivation (p33 and p26 isoforms), while others function in latency (p23 and 19 isoforms). However, nothing is known about the mechanism by which UL136 isoforms function or their interplay with other UL133-UL138 proteins to regulate latency and reactivation. Intriguingly, the full-length, membrane-bound isoform of UL136 (p33) is unstable and targeted for rapid turnover (t1/2≤1h), whereas other UL136 isoforms are stable (t1/2>6h). Stabilization of p33 by specific mutation results in a virus that cannot maintain latency and replicates, indicating that its rapid turnover is critical for maintenance of latency. From these findings, we hypothesize that the UL136 isoforms modulate the UL135/UL138 switch to reinforce decisions to stably maintain or exit latency. We propose 3 aims to address this hypothesis. Aim 1 will determine how UL136 p33 is targeted for rapid turnover and the significance of its destabilization to the decision to maintain latency or to reactivate. Aim 2 will define epistatic interplay between the UL136 isoforms and with other UL133-UL138 proteins to modulate latency and reactivation. Aim 3 will identify UL136-host interacting partners to define the mechanisms by which UL136 modulates the switch between latency and reactivation. Our work reveals new directions in HCMV latency- specifically, distinct roles for each UL136 isoform in modulating decisions to maintain the latent infection (UL138-dominant) or to reactivate (UL135-dominant). Understanding the mechanisms by which UL136 isoforms modulate the tipping point between latency and reactivation will expand the mechanistic paradigms surrounding herpesvirus latency and reactivation.