HSV-1利用宿主蛋白BRD4协助自身基因转录的分子机制研究

Transcription of Herpes Simplex Virus-I (HSV-1) genes are dependent on host cell transcriptional machinery, although detailed mechanisms remain elusive. In previous studies, we identified the host factor, Bromodomain-containing protein 4 (BRD4), as a critical player in HSV-1 infection. BRD4 regulates gene transcription of HSV-1 by recruiting P-TEFb to viral gene promoters, and the release of BRD4 from host chromatin induced by JNK phosphorylation during HSV-1 infection is the key step for the virus to effectively utilize BRD4 in its gene transcription. Based on the preliminary results, we hypothesized that HSV-1 utilizes BRD4 to recruit active transcription factors like P-TEFb for its gene transcription through JNK activation. To clarify the specific mechanisms, we plan to use a series of biotechnology including gene silence/knock out, over expression and small molecular inhibitors to evaluate the influences of JNK pathway on BRD4 release from chromatin during infection. Co-IP，and ChIP assays combined with Mass spectrometry analysis will be performed to search for the proteins that interact with JNK and BRD4 during transcription of viral genes, and analyze their function in the processes of JNK activation and BRD4 release. This research is intended to elucidate the mechanism of how the virus can effectively use BRD4 to drive and facilitate its own gene transcription through JNK pathway, and to clarify the important roles of JNK and BRD4 in transcriptional regulatory networks of HSV-1 genes.

我们前期研究发现：BRD4募集P-TEFb至HSV-1基因启动子区，是HSV-1基因转录必须依赖并利用的宿主转录系统中的关键蛋白，而HSV-1通过JNK磷酸化引起BRD4与宿主染色质解离是其有效利用BRD4协助自身基因转录的关键。为探明“HSV-1活化JNK通路竞争性利用BRD4募集胞内活性转录因子协助自身基因转录”的分子机制，本项目拟在HSV-1溶细胞感染的细胞模型上运用基因沉默/敲除、过表达及小分子抑制剂处理等手段评估JNK及相关通路蛋白的变化及其对BRD4与染色质解离的影响；应用Co-IP、ChIP、质谱等方法进一步捕获、鉴定与JNK、BRD4相互作用及与病毒启动子区结合的病毒/宿主蛋白，分析其在JNK活化致BRD4与染色质解离及BRD4募集P-TEFb中的作用，最终阐明HSV-1如何利用BRD4协助其基因转录的具体机制，明确JNK、BRD4在HSV-1基因转录调控网络中的重要作用。

本课题基于前期研究基础，聚焦于HSV-1基因转录调控过程继续深入分析,发现并证实了HSV-1感染引起JNK磷酸化致BRD4与宿主染色质解离是其有效利用BRD4协助自身基因转录的关键。从分子水平验证了JNK及相关通路蛋白的变化及其对BRD4与染色质解离影响，发现了HSV-1活化JNK通路引起BRD4与染色质解离的关键因素及其具体功能，这部分的工作正在投稿中；通过免疫共沉淀及蛋白质谱分析等方法进一步发现了与JNK、BRD4相互作用并可能参与介导BRD4-P-TEFb-RNAP II转录复合物与病毒启动子区结合并协助其基因转录的病毒蛋白ICP27，并对其功能进行了初步验证，但对其功能及作用机制的解析工作仍在持续开展中。本研究初步揭示JNK、BRD4为基础调控HSV-1基因转录的分子网络，基本阐明了HSV-1通过活化JNK通路有效利用BRD4协助其基因转录的分子机制。为进一步揭示转录过程中HSV-1与宿主间复杂相互作用及深入研究、理解HSV-1复杂感染机理的形成机制提供了重要实验依据。