Mechanisms of Human Cytomegalovirus Latency in Primary Human Hematopoietic Cells

项目总结 人巨细胞病毒是一种在宿主体内建立终生潜伏感染的β疱疹病毒。 从潜伏期重新激活可能会导致免疫缺陷个体患上危及生命的疾病，尤其是 干细胞和实体器官移植接受者。我们的研究计划长期以来一直专注于定义 HCMV决定因素和宿主相互作用是潜伏期和重新激活的机械性核心。在以前的 在资助期内，我们在巨细胞病毒基因组的ULb‘区域定义了一个跨越 基因UL133-UL138。在这个区域内，我们已经确定了病毒编码的抑制子和病毒的激活子 复制。UL138抑制复制以获得延迟，而UL135激活复制以重新激活 部分，通过克服UL138的压制作用。UL135和UL138以相反的作用调节EGFR 并且共同包括通过调节宿主信号来控制病毒潜伏期和重新激活的开关。最近， UL136已经成为这一开关的关键调节器。UL136编码5种交替的蛋白质形式或异构体 (p33、p26、p25、p23和p19，基于它们的分子质量)，仅在它们的N-末端序列上不同。 在生殖性感染中，UL136亚型相对于UL135和UL138在较晚的时间积累，需要 启动人巨细胞病毒基因组合成以获得最大表达。某些UL136亚型是必需的 重新激活(p33和p26亚型)，而其他功能以潜伏期(p23和19亚型)起作用。然而，什么都没有 已知UL136亚型的作用机制或它们与其他UL133-UL138的相互作用 调节潜伏期和重新激活的蛋白质。有趣的是，UL136的全长、膜结合的亚型 (P33)是不稳定的，目标是快速周转(T1/2≤1h)，而其他UL136亚型是稳定的(T1/2&gt；6h)。 通过特定突变稳定p33会导致病毒不能维持潜伏期和复制，这表明 它的快速周转对于保持延迟至关重要。根据这些发现，我们假设 UL136亚型调节UL135/UL138开关，以加强稳定维持或退出延迟的决策。我们 提案3旨在解决这一假设。目标1将确定UL136 p33如何针对快速更替 以及它的不稳定对维持潜伏期或重新激活的决定的重要性。目标2将定义 UL136亚型之间的上位性相互作用以及与其他UL133-UL138蛋白的上位性相互作用，以调节潜伏期和 重新激活。AIM 3将确定UL136-主机交互合作伙伴，以定义UL136 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- 具体地说，每个UL136亚型在调节维持潜伏感染的决策中扮演着不同的角色 (UL138-主导)或重新激活(UL135-主导)。了解UL136通过哪些机制 异构体调节潜伏期和重新激活之间的临界点将扩展机械范式 周围的疱疹病毒潜伏期和重新激活。

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.