Human Cytomegalovirus US28 Ligand Binding Activity Is Required for Latency in CD34+ Hematopoietic Progenitor Cells and Humanized NSG Mice
Human Cytomegalovirus US28 Ligand Binding Activity Is Required for Latency in CD34+ Hematopoietic Progenitor Cells and Humanized NSG Mice
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DOI：
10.1128/mbio.01889-19
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发表时间：
2019-07-01
期刊：
影响因子：
6.4
通讯作者：
Streblow, Daniel N.
中科院分区：
文献类型：
--
作者：
Crawford, Lindsey B.;Caposio, Patrizia;Streblow, Daniel N.
人巨细胞病毒（HCMV）感染CD34（+）造血祖细胞（CD34(+) HPCs）为干细胞移植患者提供了一个关键的病毒库，病毒再激活仍然是发病率和死亡率的重要原因。HCMV趋化因子受体US28参与病毒潜伏期和再激活的调控。为了探讨US28信号在潜伏期和再激活中的作用，我们分析了表达US28的CD34(+) HPCs中的蛋白酪氨酸激酶信号。CD34(+) HPCs中的us28配体信号传导诱导了细胞激活和分化的关键调节因子的变化。利用CD34(+) HPCs进行的体外潜伏期和再激活试验表明，US28是病毒再激活所必需的，而不是潜伏期的建立或维持。同样地，人源化NSG小鼠（huNSG）感染TB40E-GFP-US28stop后，在粒细胞集落刺激因子治疗后未能重新激活，但病毒基因组水平保持不变。有趣的是，hcmv介导的体内和体外潜伏期造血变化也依赖于US28，因为US28直接促进向髓系分化。为了确定US28的构成活性和/或配体结合活性是否需要潜伏期和再激活，我们在体外用含有US28- r129a突变（无CA）或Y16F突变（无配体结合）的HCMV TB40E-GFP感染huNSG小鼠和CD34(+) HPCs。TB40E-GFP-US28-R129A在潜伏期维持正常，表现出正常的再激活动力学。相比之下，TB40E-GFP-US28-Y16F在潜伏期和再激活期间表现出高水平的病毒基因组，表明病毒没有建立潜伏期。这些数据表明，US28是病毒再激活所必需的，而配体结合活性是病毒潜伏期所必需的，突出了US28在HCMV潜伏期和再激活中的复杂作用。人巨细胞病毒（HCMV）可以在感染CD34（+）造血祖细胞（HPCs）后建立潜伏期，而潜伏期的再激活是骨髓和实体器官移植患者病毒性疾病和加速移植物衰竭的重要原因。HCMV感染HPCs的确切分子机制尚不明确；然而，已知选择的病毒基因产物调节潜伏期和再激活方面。hcmv编码的趋化因子受体US28结合多种CC趋化因子和CX3CR1，在病毒生命周期的潜伏期和裂解期均表达，并在潜伏期和再激活中发挥作用。然而，US28表达的具体时间和配体结合在这些过程中的作用尚不清楚。在本报告中，我们确定US28用于重新激活，但不用于维持延迟。然而，当潜伏期存在时，US28配体结合活性对于维持病毒处于静止状态至关重要。我们将潜伏期和再激活的调节归因于US28在促进髓系细胞分化中的作用。这些数据强调了US28在HCMV潜伏期和再激活期间的动态和多功能特性。
Human cytomegalovirus (HCMV) infection of CD34(+) hematopoietic progenitor cells (CD34(+) HPCs) provides a critical reservoir of virus in stem cell transplant patients, and viral reactivation remains a significant cause of morbidity and mortality. The HCMV chemokine receptor US28 is implicated in the regulation of viral latency and reactivation. To explore the role of US28 signaling in latency and reactivation, we analyzed protein tyrosine kinase signaling in CD34(+) HPCs expressing US28. US28-ligand signaling in CD34(+) HPCs induced changes in key regulators of cellular activation and differentiation. In vitro latency and reactivation assays utilizing CD34(+) HPCs indicated that US28 was required for viral reactivation but not latency establishment or maintenance. Similarly, humanized NSG mice (huNSG) infected with TB40E-GFP-US28stop failed to reactivate upon treatment with granulocyte-colony-stimulating factor, but viral genome levels were maintained. Interestingly, HCMV-mediated changes in hematopoiesis during latency in vivo and in vitro was also dependent upon US28, as US28 directly promoted differentiation toward the myeloid lineage. To determine whether US28 constitutive activity and/or ligand-binding activity were required for latency and reactivation, we infected both huNSG mice and CD34(+) HPCs in vitro with HCMV TB40E-GFP containing the US28-R129A mutation (no CA) or Y16F mutation (no ligand binding). TB40E-GFP-US28-R129A was maintained during latency and exhibited normal reactivation kinetics. In contrast, TB40E-GFP-US28-Y16F exhibited high levels of viral genome during latency and reactivation, indicating that the virus did not establish latency. These data indicate that US28 is necessary for viral reactivation and ligand binding activity is required for viral latency, highlighting the complex role of US28 during HCMV latency and reactivation.IMPORTANCE Human cytomegalovirus (HCMV) can establish latency following infection of CD34(+) hematopoietic progenitor cells (HPCs), and reactivation from latency is a significant cause of viral disease and accelerated graft failure in bone marrow and solid-organ transplant patients. The precise molecular mechanisms of HCMV infection in HPCs are not well defined; however, select viral gene products are known to regulate aspects of latency and reactivation. The HCMV-encoded chemokine receptor US28, which binds multiple CC chemokines as well as CX3CR1, is expressed both during latent and lytic phases of the virus life cycle and plays a role in latency and reactivation. However, the specific timing of US28 expression and the role of ligand binding in these processes are not well defined. In this report, we determined that US28 is required for reactivation but not for maintaining latency. However, when present during latency, US28 ligand binding activity is critical to maintaining the virus in a quiescent state. We attribute the regulation of both latency and reactivation to the role of US28 in promoting myeloid lineage cell differentiation. These data highlight the dynamic and multifunctional nature of US28 during HCMV latency and reactivation.