HCMV US28 regulation of host cell signaling in viral latency and hematopoiesis

项目3摘要 人巨细胞病毒是一种疱疹病毒，感染44%-100%的人，至今仍是一种 实体器官移植和异基因造血干细胞致病和死亡的重要原因 细胞移植(SCT)接受者。SCT患者的感染通常与骨髓抑制和移植物有关。 失败是由于病毒从潜伏期重新激活，但相关机制在很大程度上仍不清楚。巨细胞病毒 编码多种潜伏期相关基因产物，包括趋化因子受体US28，它与CC- 趋化因子以及CX3C-chmokine Fractalkine。US28信号在配体非依赖性和配体非依赖性中都存在 依赖的方式，但下游信号通路和后果是独一无二的。 重要的是，我们在这里证明了在体外CD34+的潜伏期和/或重新激活都需要US28 造血祖细胞(HPC)以及人源化的小鼠模型。此外，我们还展示了 US28足以促进CD34+HPC向髓系细胞分化。因此，基于我们的 令人兴奋的新发现，我们假设在潜伏期US28不依赖配体的信号有助于维持 潜伏期和配体特异性信号促进CD34+HPC分化为重新激活/复制 有能力的髓系细胞，以确保病毒在适当的条件下复制，包括炎症。 US28信号也可能通过充当促进迁移的生物传感器而导致延迟的其他方面 对炎性趋化因子的作用，可能是通过增加早期基因的即刻表达 重新激活。定义US28在延迟和重新激活中的角色，确定US28信令如何交叉 EGFR信号，以及了解US28信号如何影响其他HCMV蛋白(项目1和 4)和在延迟和操作EGFR信号(项目5)期间表达的miRNAs(项目2)是 这项提案的目标。我们提出了以下具体目标：1)确定US28信号在CD34+中是如何 使用多组学方法将HPC与EGFR信号通路相交；2)确定 US28通过体外和体内CD34+HPC介导人巨细胞病毒潜伏和激活的特性 人源化BLT小鼠模型；3)确定哪些US28信号通路促进CD34+HPC 也可以使用体外CD34+HPC和体内huBLT模型进行造血。这项研究的结果将 产生新的病毒潜伏期和重新激活范例，促进开发新的治疗方法 防止病毒再激活和治疗巨细胞病毒介导的骨髓抑制。

PROJECT 3 SUMMARY Human cytomegalovirus (HCMV) is a -herpesvirus infecting 44-100% of the population and remains a significant cause of morbidity and mortality in solid organ transplant (SOT) and allogeneic hematopoietic stem cell transplant (SCT) recipients. Infection in SCT patients is often associated with myelosuppression and graft failure due to virus reactivation from latency, but the associated mechanisms are still largely unknown. HCMV encodes multiple latency-associated gene products including the chemokine receptor US28, which binds CC- chemokines as well as the CX3C-chmokine Fractalkine. US28 signals in both ligand-independent and ligand- dependent manners, but the downstream signaling pathways and consequences are unique for each. Importantly, herein we demonstrate that US28 is required for latency and/or reactivation in both in vitro CD34+ hematopoietic progenitor cells (HPCs) as well as in a humanized mouse model. In addition, we show that US28 is sufficient to promote CD34+ HPC differentiation into myeloid lineage cells. Therefore, based upon our exciting new findings, we hypothesize that US28 ligand-independent signaling during latency helps to maintain latency and that ligand-specific signaling promotes CD34+ HPC differentiation into a reactivation/replication competent myeloid lineage cell to ensure virus replication under appropriate conditions including inflammation. US28 signaling may also contribute to other aspects of latency by acting as a biosensor to promote migration towards inflammatory chemokines, and possibly by increasing immediate early gene expression during reactivation. Defining the role of US28 in latency and reactivation, determining how US28 signaling intersects with EGFR signaling, and understanding how US28 signaling influences other HCMV proteins (Projects 1 and 4) and miRNAs (Project 2) expressed during latency and manipulating EGFR signaling (Project 5) are the goals of this proposal. We propose the following specific aims: 1) To determine how US28 signaling in CD34+ HPCs intersects with EGFR signaling pathways using a multi-omics approach; 2) To determine what molecular characteristics of US28 mediate HCMV latency and reactivation using both in vitro CD34+ HPCs and in vivo humanized BLT mouse models; 3) To determine what US28 signaling pathways promote CD34+ HPC hematopoiesis also using both in vitro CD34+ HPC and in vivo huBLT models. Results of this study will generate new virus latency and reactivation paradigms promoting the development of novel therapies to prevent virus reactivation and to treat HCMV-mediated myelosuppression.