胶质瘤特异性转录因子ATF5通过调控HCMV IE表达影响病毒复制的机制

Several reports have identified the presence of human cytomegalovirus (HCMV) gene products endogenously expressed in glioma tissue and cells, with a long-term and low-level infection and a subset of viral genes being consistently expressed. The viral infection pattern and replication level depend largely on the immediate-early gene (IE) in host cells. Our previous work found that HCMV replication levels were different in glioma cell lines with various degrees of endogenous ATF5. Several ATF5 consensuses DNA binding sequences are found in HCMV major immediate early promoter (MIEP) by bioinformatics prediction. Our previous works indicate that histone H3 at MIEP may be acetylated through ATF5/p300 complex. This research will focus on the mechanism of HCMV replication level in glioma cell lines where endogenous ATF5 expression is different. Firstly the HCMV IE, E, L gene, ATF5 expression and H3K9/K14 acetylation will be detected in paraffin sections of surgically excised glioma by immunohistochemistry staining. Then the activation of HCMV IE resulting in upregulating of HCMV replication via ATF5 binding to the consensuses DNA binding sequences in HCMV IE promoter-regulatory region will be studied. The H3K9/K14 acetylation level of MIEP region after ATF5 binding will be detected too. After that, mice models bearing glioma cells will be established to verify the ATF5 function on regulating HCMV replication in vivo. This research is trying to partly clarify the molecular mechanism between HCMV infection states and transcription factors environment in host cells.

人巨细胞病毒（HCMV）能以一种低水平的持续性感染状态存在于神经胶质瘤细胞内，具体机制尚不清楚。HCMV即刻早期基因（IE）是病毒依靠宿主细胞的转录因子环境而被激活的第一个基因，其表达程度制约着病毒的复制水平。我们前期研究发现，在转录因子ATF5表达水平不同的胶质瘤细胞系中，HCMV的复制水平存在差异。ATF5是一个与胶质瘤密切相关的转录因子，能与乙酰化酶p300等多种蛋白相互作用调节基因转录。生物信息学分析预示，ATF5能够与IE基因启动子（MIEP）区结合。本项目拟以胶质瘤临床标本中ATF5表达与HCMV复制水平相关性为切入点；利用HCMV复制水平不同的胶质瘤细胞系为模型，阐明ATF5与MIEP结合后，通过募集乙酰化酶，改变MIEP区组蛋白乙酰化水平调控IE基因表达，进而影响HCMV复制水平的分子机制。研究为揭示HCMV利用宿主细胞转录因子环境，建立低水平复制的机制提供理论依据。

人巨细胞病毒（Human Cytomegalovirus ，HCMV）以潜伏感染为主要特征。研究发现HCMV潜伏感染与神经胶质瘤关系密切，病毒可以一种低水平的持续性感染存在于胶质瘤细胞内。我们之前的研究已揭示了其促进胶质瘤细胞增殖的机制，为了充分研究HCMV与宿主细胞相互作用的关系，本项目着眼于宿主细胞内转录因子环境对HCMV复制的影响。HCMV即刻早期基因（IE）的启动子（MIEP）是病毒单纯依靠宿主细胞的转录因子环境而被激活的第一个启动子，其表达程度制约着病毒在宿主细胞内的复制水平。启动子区预测我们发现，转录因子ATF5能够与MIEP启动子区结合。通过胶质瘤临床标本的免疫组化和免疫荧光检测我们确证了HCMV IE蛋白与ATF5表达相关性。细胞模型上，我们进一步确证了ATF5表达水平与HCMV复制水平的关系，并通过荧光素梅报告基因、EMSA及ChIP等技术，证实了ATF5能与MIEP启动子区特异性的位点结合。在研究过程中，我们还发现了miR-141-3p对ATF5的调控作用，进一步丰富了ATF5与HCMV相互作用，调节胶质瘤进程的理论基础。