HCMV UL7 regulation of host cell signaling in viral latency and hematopoiesis

项目4：总结 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。 由于病毒从潜伏期重新激活而导致的失败，但相关的机制在很大程度上仍不清楚。我们有 最近发现了一种分泌型病毒因子UL7，它含有一个具有结构和功能的Ig样结构域 与癌胚抗原相关细胞黏附分子1(CEACAM1)同源，CEACAM1是一种具有多种 包括分化在内的细胞活动。我们最近证明UL7刺激炎性细胞 内皮细胞的反应，诱导IL-6和其他细胞因子的分泌，部分是通过 JAK/STAT和MAP激酶通路中STAT3和ERK1/2的磷酸化。IL-6和IL-6的分泌 据报道，ERK-MAPK信号通路的激活可以刺激病毒的重新激活。重要的是 我们已经证明，尽管UL7在潜伏期内没有表达，但缺失UL7的HCMV未能 在体外和体内潜伏感染的CD34+HPC中重新激活，尽管病毒具有复制能力 成纤维细胞。这些数据表明，UL7在潜伏感染的CD34+中启动重新激活后起作用 HPC，但在裂解程序启动之前，这表明细胞的UL7重新编程对于 重新激活事件。此外，我们还发现UL7足以促进CD34+HPC的分化 转化为髓系细胞。因此，根据我们令人兴奋的新发现，我们假设UL7 调节表皮生长因子受体(EGFR)下游的JAK-STAT-AKT通路以 促进病毒传播的潜伏期和细胞分化的重新激活。在这个项目中，我们将 检测CD34+HPC中的UL7信号并确定病毒重新激活和 在感染的背景下的造血。我们还将确定这些UL7路径是否与 受US28(项目3)和UL133/8(项目1)或HCMV miRNAs(项目2)、其他HCMV调节的通路 调节EGFR下游主机信号的潜伏期和差异性的重要因素(项目5)。 我们提出了以下具体目标：1)确定CD34+HPC中的UL7信号如何与 用多组学方法研究EGFR信号转导途径；2)确定 UL7体外CD34+人造血祖细胞和体内人源化BLT介导人巨细胞病毒潜伏和再激活 小鼠模型；3)确定哪些UL7信号通路促进CD34+HPC的造血 CD34+HPC体外模型和活体huBLT模型。这项研究的结果将产生新的病毒潜伏期和 重新激活范例促进了预防病毒重新激活和治疗的新疗法的发展 巨噬细胞病毒介导的骨髓抑制。

PROJECT 4: 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. We have recently identified a secreted viral factor, UL7, containing an Ig-like domain with structural and functional homology to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a protein with multiple cellular activities including differentiation. We recently demonstrated that UL7 stimulates an inflammatory response in endothelial cells, inducing the secretion of IL-6 and other cytokines mediated in part through the phosphorylation of STAT3 and ERK1/2 in the JAK/STAT and MAP kinase pathways. Secretion of IL-6 and activation of the ERK-MAPK signaling pathway have been reported to stimulate viral reactivation. Importantly, we have shown that although UL7 is not expressed during latency, HCMV with a deletion of UL7 fails to reactivate in latently infected CD34+ HPCs in vitro and in vivo although the virus is replication competent in fibroblasts. These data indicate that UL7 is acting after the initiation of reactivation in latently infected CD34+ HPCs but before the lytic program is initiated, suggesting that UL7 reprogramming of the cell is essential for the reactivation event. In addition, we have shown that UL7 is sufficient to promote CD34+ HPC differentiation into myeloid lineage cells. Therefore, based upon our exciting new findings, we hypothesize that UL7 modulates the JAK-STAT-AKT pathways downstream of the Epidermal Growth Factor Receptor (EGFR) to promote reactivation from latency and cellular differentiation for viral dissemination. In this project we will examine UL7 signaling in CD34+ HPCs and identify the cellular pathways necessary for viral reactivation and hematopoiesis in the context of infection. We will also determine whether these UL7 pathways cross-talk with pathways modulated by US28 (Project 3) and UL133/8 (Project 1) or HCMV miRNAs (Project 2), other HCMV factors important to latency and differentiation that modulate host signaling downstream of EGFR (Project 5). We propose the following specific aims: 1) To determine how UL7 signaling in CD34+ HPCs intersects with EGFR signaling pathways using a multi-omics approach; 2) To determine what molecular characteristics of UL7 mediate HCMV latency and reactivation using both in vitro CD34+ HPCs and in vivo humanized BLT mouse model; 3) To determine what UL7 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.