lncRNA4.9介导的NAB2对EGR1-IE2的调控在人巨细胞病毒潜伏感染中的作用及其机制

Human cytomegalovirus (HCMV) is the most common cause of perinatal infections. Recent study has shown that long noncoding (lnc) RNA4.9 encoded by HCMV may be closely related to viral latent infection, but the mechanisms remain poorly understood. In preliminary experiment, we predicted and verified lncRNA4.9 could up-regulate the expression of early growth response gene 1 (EGR1) by targeted inhibition on NGFI-A binding protein 2 (NAB2). In this study, a previously constructed model of HCMV latency by infecting CD14+ monocyte will be used to explore the effects of lncRNA4.9 on HCMV latency and the regulatory mechanisms. Firstly, basing on the respective regulation of lncRNA4.9 and NAB2 levels, the expressions of lncRNA4.9, NAB2, EGR1 would be detected, the changes of HCMV latent state would be monitored, and the specific protein combinating to EGR1 would also be immune co-precipitated and mass spectrometry analyzed to reveal the effect of lncRNA4.9 on HCMV latency by regulating EGR1-IE2. Secondly, the changes of cell cycle would be detected after the respective regulation on the expressions of lncRNA4.9 and TGF-β. Furthermore, the levels of immune factors and T cell activation would be detected, and the changes of HCMV latent state would be monitored in a co-culture model of HCMV latently infected cells with T lymphocytes to identify the effect of lncRNA4.9 on HCMV latency by regulating TGF-β to inhibit cell cycle and T cell functions. Finally, basing on the co-culture model of HCMV latently infected cells with inhibited/activated T lymphocytes, the levels of immune factors and T cell activation would be detected, and the lncRNA4.9 expression and the changes of HCMV latent state would be monitored to interpret the effect of immune activation on HCMV latency. This study will be helpful to provide novel ideas on the pathogenesis of HCMV latent infection and new targets of drug development for HCMV treatment and prevention.

HCMV是围产期感染的常见病原体，最新研究表明其编码的lncRNA4.9与潜伏感染密切相关，但机制未明。我们前期工作已证实lncRNA4.9通过抑制NAB2而上调EGR1的表达。在此基础上，本项目以HCMV潜伏感染的CD14+细胞为研究模型，首先分别调控lncRNA4.9和NAB2的表达，监测病毒潜伏状态，免疫共沉淀和质谱分析EGR1的结合蛋白，探明lncRNA4.9通过调控EGR1-IE2对HCMV潜伏感染的作用；然后分别调控lncRNA4.9和TGF-β的表达，检测细胞周期变化，进一步与T细胞共培养后检测免疫因子和T细胞活化水平，明确lncRNA4.9通过调控TGF-β对HCMV潜伏感染的影响；最后，抑制/激活T细胞并与潜伏感染细胞共培养，监测病毒潜伏状态，揭示免疫激活对HCMV潜伏感染的影响。本项目将为阐明HCMV潜伏感染的发病机制提供新思路，为HCMV防治药物的研发提供新靶标。

人巨细胞病毒（HCMV）编码的lncRNA4.9与其潜伏感染密切相关，可能通过抑制NAB2上调EGR1的表达。本项目是在构建HCMV潜伏感染模型的基础上，首先阐明lncRNA4.9通过调控EGR1-IE2对HCMV潜伏感染的作用，然后明确lncRNA4.9通过调控TGF-β对HCMV潜伏感染的影响，最后揭示T细胞免疫状态对HCMV潜伏感染细胞中病毒复制的影响。主要取得以下研究结果：⑴ 成功构建稳定的HCMV潜伏感染模型；⑵ 明确lncRNA4.9通过调控EGR1-IE2促进HCMV潜伏感染；⑶ 明确lncRNA4.9通过调控TGF-β促进HCMV潜伏感染；⑷ 明确T细胞激活能抑制HCMV潜伏感染细胞中病毒的复制。本项目已如期完成所有研究计划要点，发表SCI收录论文2篇，中华系列期刊论文2篇，申请发明专利2项，培养硕士研究生2名。本项目阐明的lncRNA4.9对HCMV潜伏感染的调控及其机制将为深入探索HCMV潜伏感染的致病机制提供新思路，为HCMV防治药物和疫苗的研发提供新靶标。