Mechanisms of Human Cytomegalovirus Latency in Primary Human Hematopoietic Cells

项目摘要 人巨细胞病毒（HCMV）是一种β-疱疹病毒，在其宿主中建立终身潜伏感染。 潜伏期的再激活可导致免疫缺陷个体发生危及生命的疾病， 干细胞和实体器官移植受者。我们的研究计划长期以来一直专注于定义 HCMV决定簇和宿主相互作用是潜伏期和再激活的机制核心。前几 在资助期间，我们在HCMV基因组的ULb'区域内定义了一个多顺反子基因座，该基因座跨越了 基因UL 133-UL 138。在这一区域内，我们已经鉴定出病毒编码的抑制因子和病毒激活因子。 复制的UL 138抑制复制的潜伏期，而UL 135激活复制的再激活， 第一，克服UL 138的压制作用。UL 135和UL 138以相反的作用调节EGFR 并且一起构成通过调节宿主信号传导来控制病毒潜伏期和再活化的开关。最近， UL 136已成为该开关的关键调制器。UL 136编码5种交替的蛋白质形式或同种型 (p33，p26，p25，p23和p19，基于它们的分子量），它们仅在它们的N-末端序列上不同。 在生产性感染中，UL 136同种型相对于UL 135和UL 138在较晚的时间积累，这需要 HCMV基因组合成的开始，以达到最大表达。需要一些UL 136亚型 再激活（p33和p26同种型），而其它的在潜伏期起作用（p23和19同种型）。然而， 关于UL 136同种型发挥作用的机制或它们与其它UL 133-UL 138的相互作用是已知的 调节潜伏期和再激活的蛋白质。有趣的是，UL 136的全长膜结合亚型 (p33)UL 136是不稳定的，目标是快速转换（t1/2≤1h），而其他UL 136亚型是稳定的（t1/2> 6 h）。 通过特定突变稳定p33导致病毒不能维持潜伏期和复制，表明 其快速周转对于保持延迟至关重要。根据这些发现，我们假设 UL 136同种型调节UL 135/UL 138开关，以加强稳定维持或退出延迟的决定。我们 建议3旨在解决这一假设。目标1将确定UL 136 p33如何靶向快速周转 以及它的不稳定对决定保持潜伏期或重新激活的重要性。目标2将定义 UL 136同种型之间以及与其它UL 133-UL 138蛋白之间的上位相互作用以调节潜伏期， 重新激活目标3将确定UL 136-宿主相互作用伙伴，以定义UL 136 调节延迟和重新激活之间的切换。我们的工作揭示了HCMV潜伏期的新方向- 具体而言，每种UL 136亚型在调节维持潜伏感染的决定中的不同作用 （UL 138-显性）或重新激活（UL 135-显性）。了解UL 136的机制 同种型调节潜伏期和再激活之间的临界点将扩展机制范例 周围的疱疹病毒潜伏期和再激活。

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.