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

项目2项目摘要/摘要 疱疹病毒miRNAs针对参与病毒免疫识别的许多不同的细胞和病毒过程， 细胞凋亡、细胞周期调控、细胞运输以及病毒潜伏和裂解复制。我们已经做出了 阐明人巨细胞病毒miRNAs功能作用的重要进展，包括鉴定人巨细胞病毒 调节HCMV IE1以及参与细胞周期调控、形成的多个细胞基因的miRNAs 病毒组装复合体和TLR2信号。重要的是，我们已经观察到HCMV miRNAs 协同工作，有效下调单个基因，并使HCMV miRNAs靶向多个 同一细胞通路中的单个基因。最近，我们确定了几个HCMV miRNAs 靶向表皮生长因子受体(EGFR)信号通路的多个成员 在病毒潜伏期和造血方面很重要。我们已经观察到两个人巨细胞病毒的双重突变 针对这一途径的miRNA导致病毒重新激活的增加，而另外两种病毒miRNA加倍 突变体在体外的人类祖细胞(HPC)培养系统中无法重新激活。最后，EGFR信号具有 也被证明在HPC的造血中很重要。我们观察到单核细胞感染HPC 双重HCMV miRNA突变导致CFU-GM、CFU-GEMM和BFU-E集落水平降低 与WT HCMV相比，miRNAs在形成和骨髓抑制中的作用 造血术。因此，我们推测，hcmv miRNA对egfr信号的调节起着关键作用。 在病毒潜伏和重新激活以及造血方面。在当前的提案中，我们将描述 EGFR、HCMV、miRNA靶标和EGFR miRNA靶标与病毒潜伏期和 CD34-HPC体外复制。这些研究将扩展到新开发的人类胎儿骨骼--肝脏 和胸腺(BLT)nod-cerdIL2Rgc缺失型小鼠模型，能够支持潜伏的HCMV感染以及 从延迟重新激活(核心A)。我们将确定针对这一途径的HCMV miRNAs 在项目1中，在建立和维持病毒潜伏期的过程中与UL133/8 EGFR调节相交。 此外，几个HCMV miRNAs还针对由HCMV潜伏期基因US28和 UL7。我们将研究这些miRNAs在调节US28信号和US28-EGFR信号中所起的作用 项目3和UL7(项目4)中的串扰。最后，我们将确定HCMV EGFR的贡献 造血中的miRNAs与项目5中的WT HCMV结果的比较。

PROJECT 2 PROJECT SUMMARY/ABSTRACT Herpesvirus miRNAs target many different cellular and viral processes involved in viral immune recognition, apoptosis, cell cycle regulation, cellular trafficking as well as viral latency and lytic replication. We have made significant advances elucidating functional roles for HCMV miRNAs including the identification of HCMV miRNAs that regulate HCMV IE1 as well as multiple cellular genes involved in cell cycle modulation, formation of the viral assembly complex, and TLR2 signaling. Importantly, we have observed that the HCMV miRNAs work coordinately to efficiently down-regulate individual genes and that the HCMV miRNAs target multiple individual genes within the same cellular pathways. Recently, we have determined that several HCMV miRNAs target multiple members of the Epidermal Growth Factor Receptor (EGFR) signaling pathways that are important in viral latency and hematopoiesis. We have observed that a double HCMV mutation of two of the miRNAs targeting this pathway results in an increase in viral reactivation while two other viral miRNA double mutants fail to reactivate in a human progenitor cell (HPC) culture system in vitro. Lastly, EGFR signaling has also been shown to be important in HPC hematopoiesis. We have observed that infection of HPCs with single and double HCMV miRNA mutations results in reduced levels of CFU-GM, CFU-GEMM and BFU-E colony formation as well as myelosuppression in comparison to WT HCMV, suggesting a role for the miRNAs in hematopoiesis. Therefore, we hypothesize that HCMV miRNA regulation of EGFR signaling plays a critical role in viral latency and reactivation as well as hematopoiesis. In the current proposal we will characterize the EGFR HCMV miRNA targetome and the functional relevance of the EGFR miRNA targets for viral latency and replication in CD34+ HPC in vitro. These studies will be extended in a newly developed human fetal bone, liver and thymus (BLT) NOD-scidIL2Rgc null mouse model that is able to support latent HCMV infection as well as reactivation from latency (Core A). We will determine how the HCMV miRNAs that target this pathway interphase with UL133/8 EGFR regulation in the establishment and maintenance of viral latency in Project 1. Additionally, several of the HCMV miRNAs also target genes activated by the HCMV latency gene US28 and UL7. We will examine the role that these miRNAs play in regulating US28 signaling and US28-EGFR signaling cross-talk in Project 3 and UL7 (Project 4). Lastly, we will determine the contribution of the HCMV EGFR miRNAs in hematopoiesis in comparison to results of WT HCMV in Project 5.