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

摘要--项目2 人巨细胞病毒(HCMV)潜伏期重新激活在以下情况下仍是一个严重的并发症 免疫抑制。潜伏期和重新激活是受病毒蛋白子集调控的复杂过程 CD34+造血祖细胞中的非编码RNA。该项目的目标是定义如何 HCMV microRNAs(MiRNAs)提纯和塑造病毒蛋白介导的信号转导 退出延迟。我们先前已经确定，HCMV miRNAs在潜伏期干扰了多条通路- 感染细胞，包括转化生长因子、蛋白酪氨酸激酶/ERK和RhoA，对巨细胞病毒潜伏期有重要影响。重要的是 我们已经发现了HCMV miRNAs在与病毒蛋白的协调和对抗中发挥的作用。为 例如，为了在感染CD34+HPC后早期调节细胞凋亡，miR-US5-1和miR-UL112-3p起作用 与UL7(项目4)协调，以减少FOXO3a的水平和活性。相比之下，HCMV miR-US5-2和 病毒关键的EGFR下游MEK/ERK信号通路的MIR-US22靶成分 重新激活并减弱US28和UL138介导的信号。 我们已经确定RhoA是HCMV miR-US25-1的靶标，它在调节人巨细胞病毒的增殖方面发挥关键作用。 通过调节胞质分裂潜伏感染CD34+HPC。耐人寻味的是，HCMV US28(项目3)被激活 RhoA通过配体依赖的信号转导和最近的邻近标记实验鉴定了RhoGEF和 Rho GTP酶作为US28信号体的组成部分。US28突变或RhoA的化学抑制 减少病毒的重新激活，强调了US28和Rho信号在重新激活过程中的重要性。 此外，UL8(项目4)是重新激活所必需的，并通过Wnt途径激活Rho信号。 此外，邻近标记实验确定Wnt和Rho途径组件是UL8的一部分 信号体。我们已经确定了针对WNT和RHO关键组件的其他HCMV miRNAs GTP酶途径包括Wnt效应子DVL2、RhoGEF、GTPase CDC42和下游效应子 蛋白质，这表明Rho GTPase信号除了受病毒miRNAs的调控外，还受到病毒miRNAs的复杂调控 受到病毒蛋白的刺激。我们假设HCMV miRNAs靶向MEK/ERK通路的各个组成部分 在重新激活时下调病毒介导的信号，而miRNAs在潜伏期表达 靶向成分的Rho GTP酶通路对抗病毒介导的信号转导效应维持 病毒处于潜伏期。

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.