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

项目4：概要 人巨细胞病毒（HCMV）是一种疱疹病毒，感染44-100%的人群，并且仍然是一种高致病性病毒。 实体器官移植（SOT）和异基因造血干细胞移植中发病率和死亡率重要原因 细胞移植（SCT）受者。SCT患者的感染通常与骨髓抑制和移植相关。 失败是由于病毒从潜伏期重新激活，但相关的机制仍然在很大程度上未知。我们有 最近鉴定了一种分泌型病毒因子UL 7，其含有Ig样结构域， 与癌胚抗原相关细胞粘附分子1（CEACAM 1）同源，CEACAM 1是一种具有多个 包括分化在内的细胞活动。我们最近证明UL 7刺激炎症反应， 在内皮细胞的反应，诱导分泌IL-6和其他细胞因子介导的部分通过 在JAK/STAT和MAP激酶途径中STAT 3和ERK 1/2的磷酸化。IL-6的分泌和 已经报道了ERK-MAPK信号通路的激活刺激病毒再激活。重要的是， 我们已经证明，虽然UL 7在潜伏期不表达，但缺失UL 7的HCMV不能表达UL 7， 在体外和体内潜伏感染的CD 34 + HPC中重新激活，尽管病毒在体内具有复制能力， 成纤维细胞这些数据表明，UL 7在潜伏感染的CD 34+细胞中的再活化开始后起作用。 但在裂解程序启动之前，这表明细胞的UL 7重编程对于 重新激活事件。此外，我们已经表明UL 7足以促进CD 34 + HPC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UL 7 调节表皮生长因子受体（EGFR）下游的JAK-STAT-AKT通路， 促进从潜伏期的再激活和细胞分化以用于病毒传播。在这个项目中，我们将 检查CD 34 + HPC中的UL 7信号传导，并确定病毒再活化所需的细胞途径， 在感染的情况下造血。我们还将确定这些UL 7途径是否与 由US 28（项目3）和UL 133/8（项目1）或HCMV miRNA（项目2）调节的途径，其他HCMV 调节EGFR下游宿主信号传导的对潜伏期和分化重要的因子（项目5）。 我们提出了以下具体目标：1）确定CD 34 + HPC中的UL 7信号如何与CD 34 + HPCs中的UL 7信号交叉。 使用多组学方法研究EGFR信号通路; 2）确定EGFR信号通路的分子特征， 使用体外CD 34 + HPC和体内人源化BLT两者，UL 7介导HCMV潜伏期和再活化 小鼠模型; 3）为了确定什么UL 7信号通路促进CD 34 + HPC造血，也使用 体外CD 34 + HPC和体内huBLT模型。这项研究的结果将产生新的病毒潜伏期， 促进新疗法的发展，以防止病毒再活化和治疗 HCMV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.