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

摘要-项目2 人巨细胞病毒（HCMV）从潜伏期再激活仍然是一个严重的并发症， 免疫抑制潜伏期和再活化是由一组病毒蛋白调节的复杂过程， CD 34+造血祖细胞（HPC）中的非编码RNA。这个项目的目标是定义如何 HCMV microRNAs（miRNAs）改善和塑造由病毒蛋白介导的信号传导，所述病毒蛋白对于进入和 退出latency。我们先前已经确定HCMV miRNAs干扰多种途径，在潜伏期- 感染的细胞，包括TGF β 1、MEK/ERK和RhoA，它们对HCMV潜伏期具有重要意义。重要的是， 我们已经揭示了HCMV miRNA在病毒蛋白的协调和对抗中的作用。为 例如，为了在CD 34 + HPC感染后早期调节凋亡，miR-US 5 -1和miR-UL 112 - 3 p作用于 与UL 7（项目4）一致，以降低FOXO 3a的水平和活性。相反，HCMV miR-US 5 -2和 miR-US 22靶向EGFR下游的MEK/ERK信号传导途径的组分，其对病毒感染至关重要。 重新激活并减弱US 28和UL 138介导的信号传导。 我们已经确定RhoA是HCMV miR-US 25 -1的靶点，其在调节细胞增殖中起关键作用。 通过调节胞质分裂来抑制潜在感染的CD 34 + HPCs。有趣的是，HCMV US 28（项目3）激活 RhoA通过配体依赖性信号传导和最近的邻近标记实验鉴定了RhoGEFs和 Rho GTP酶作为US 28信号体的组分。US 28突变或RhoA化学抑制 减少病毒再活化，突出了US 28和Rho信号在再活化过程中的重要性。 此外，UL 8（项目4）对于再激活是必需的，并通过Wnt途径激活Rho信号传导。 此外，邻近标记实验将Wnt和Rho途径组分鉴定为UL 8的一部分。 信号体我们已经鉴定了另外的HCMV miRNAs，其靶向Wnt和Rho的关键组分， 包括Wnt效应器DVL 2、RhoGEFs、GTdR CDC 42和下游效应器的GTdR途径 蛋白质，表明Rho GT3信号转导除了被病毒miRNAs调控外， 由病毒蛋白刺激。我们假设HCMV miRNAs靶向MEK/ERK通路的组分， 在再激活时下调病毒介导的信号传导，而在潜伏期表达的miRNAs 靶向Rho GT3通路的组分，以对抗病毒介导的信号传导的作用， 潜伏期的病毒

SUMMARY - PROJECT 2 Human cytomegalovirus (HCMV) reactivation from latency remains a serious complication under conditions of immunosuppression. Latency and reactivation are complex processes regulated by a subset of viral proteins and non-coding RNAs in CD34+ hematopoietic progenitor cells (HPCs). The goal of this project is to define how HCMV microRNAs (miRNAs) refine and shape signaling mediated by viral proteins critical for the entry into and exit from latency. We have previously determined that HCMV miRNAs interfere with multiple pathways in latently- infected cells, including TGF, MEK/ERK and RhoA that have critical implications for HCMV latency. Importantly, we have uncovered a role for HCMV miRNAs acting both in coordination and in opposition to viral proteins. For example, in order to regulate apoptosis early after infection of CD34+ HPCs, miR-US5-1 and miR-UL112-3p act in concert with UL7 (Project 4) to reduce the levels and activity of FOXO3a. In contrast, HCMV miR-US5-2 and miR-US22 target components of the MEK/ERK signaling pathway downstream of EGFR critical for viral reactivation and attenuate US28- and UL138-mediated signaling. We have identified RhoA as a target of HCMV miR-US25-1 that plays a key role in regulating the proliferation of latently-infected CD34+ HPCs through modulating cytokinesis. Intriguingly, HCMV US28 (Project 3) activates RhoA through ligand-dependent signaling and recent proximity labelling experiments identified RhoGEFs and Rho GTPases as components of the US28 signalosome. Mutation of US28 or chemical inhibition of RhoA reduces viral reactivation, highlighting the importance of US28 and Rho signaling in the reactivation process. Additionally, UL8 (Project 4) is essential for reactivation and activates Rho signaling through the Wnt pathway. Moreover, proximity labelling experiments identified Wnt and Rho pathway components as part of the UL8 signalosome. We have identified additional HCMV miRNAs that target key components of the Wnt and Rho GTPase pathways including the Wnt effector DVL2, RhoGEFs, the GTPase CDC42 and downstream effector proteins, indicating that Rho GTPase signaling is intricately regulated by viral miRNAs in addition to being stimulated by viral proteins. We hypothesize that HCMV miRNAs target components of the MEK/ERK pathway to downregulate virus-mediated signaling at the time of reactivation, whereas miRNAs expressed during latency target components of the Rho GTPase pathways to oppose the effects of virus-mediated signaling to maintain the virus in latency.