人巨细胞病毒潜伏感染的自噬调控及相关IE2-Akt-Beclin 1通路的作用机制

Human cytomegalovirus (HCMV) is the most frequent cause of congenital infection. Transplacental transmission of HCMV during pregnancy or neonatal infection of premature newborns can lead to neurological damage. Both primary and latency-activation infection of HCMV during gestation pose a risk of intrauterine transmission. Given the extremely high seropositive rate in pregnant women, latent infection seems to play an important role in congenital HCMV infection. Autophagy regulation and the molecular mechanisms in HCMV latency remain poorly understood. Most of the respective viral proteins affect autophagy via binding to Beclin 1, an autophagy-related protein that constitutes part of the phosphatidylinositol-3 kinase complexes and plays a crucial role in autophagy initiation. HCMV immediate-early protein IE2 is essential for HCMV replication due to its ability to transactivate critical viral early promoters. The expression level of IE2 is accordingly regarded as the most important hallmark to discriminate productive and latent infection of HCMV. Here, we propose a new IE2-Akt-Beclin 1 signaling pathway in the autophagy regulation of HCMV latency based on the latest research progress. In this study, a model of HCMV latency by infecting CD14+ monocyte will be constructed for exploring the regulatory mechanisms of autophagy and IE2-Akt-Beclin 1 pathway. The mRNA and protein levels of several autophagy-related markers such as Beclin 1, LC3, and p62 in this model will be firstly detected to evaluate the characteristics of autophagy regulation in HCMV latency. Furthermore, the autophagy level in this model will be modulated by chemical agents or small interfering RNA (siRNA) and microRNAs targeting to Beclin 1. The susceptibility of CD14+ monocyte to HCMV or the viral infection status will be compared between before and after the modulations to determine the effect of autophagy on HCMV latent infection. For the purpose of understanding the role of IE2-Akt-Beclin 1 pathway in autophagy regulation, the expressions and phosphorylations of Akt and Beclin 1 will be detected before and after changing the level of IE2 in HCMV productive and latent infection cell models. In addition, immunoprecipitation and mass spectrometric analysis will be carried out to identify the key molecules interacting with Beclin 1 in the upstream and downstream of the IE2-Akt-Beclin 1 pathway. The autophagy regulation in HCMV latency and the role of relevant IE2-Akt-Beclin 1 pathway revealed in this study will be helpful to further explore the pathogenesis of HCMV latency and to facilitate the development of novel vaccines and drugs against this virus.

人巨细胞病毒（HCMV）是围产期感染的常见病原，近期研究表明其致病性和免疫逃避可能与自噬相关，但确切机制尚未阐明。Beclin 1是自噬过程中的关键蛋白，其作用机制和相关信号通路有待深入研究。本项目在前期工作基础上，拟建立HCMV潜伏感染CD14+细胞模型，首先检测模型中的自噬标志物，探明HCMV潜伏感染的自噬水平变化特点；进而通过改变潜伏感染模型中的自噬水平，监测HCMV细胞易感性或感染状态的变化，明确自噬水平对HCMV潜伏感染的影响；最后调控IE2水平探讨其对Akt和Beclin 1表达及磷酸化的作用，并采用免疫沉淀和质谱分析鉴定IE2-Akt-Beclin 1通路上、下游结合Beclin 1的关键蛋白，揭示IE2-Akt-Beclin 1通路在HCMV潜伏感染自噬调控中的分子机制。本项目将为阐明HCMV潜伏感染的发病机制提供新思路，并为HCMV防治药物和疫苗的研发提供新靶标。

细胞自噬在病原体感染中发挥重要功能，许多病毒与自噬的相互调控通过自噬相关蛋白Beclin 1 起作用。目前人巨细胞病毒（HCMV）感染与自噬的相关性研究均在增殖感染模型中进行。本项目是在构建HCMV潜伏感染模型的基础上，首先阐明HCMV潜伏感染对细胞自噬的影响，进而通过调控细胞自噬水平，探索细胞自噬对HCMV建立和维持潜伏感染的影响，最后揭示IE2-Akt-Beclin1通路在HCMV潜伏感染调控自噬中的作用机制。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HCMV感染促进THP-1细胞的自噬，但在建立潜伏感染后，促进细胞自噬强度较未建立潜伏感染时减弱；⑶ 明确自噬调控药物3-甲基丙氨酸和雷帕霉素改变THP-1细胞的自噬水平对HCMV进入或维持潜伏感染状态均无影响；⑷ 明确抑制Beclin1表达从而改变THP-1细胞的自噬水平对HCMV进入或维持潜伏感染状态均无影响；⑸ 明确HCMV IE2通过Akt正向调控潜伏感染细胞的自噬水平。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，国内核心期刊论著1篇和综述1篇，申请发明专利2项，培养硕士研究生3名。本项目阐明的HCMV潜伏感染的自噬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。

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