Brd4通过一种新结合蛋白TDP-43调节HIV-1转录的机制研究

Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndromes (AIDS). Although great progress has been made in eliminating productive HIV infection in patients, a small fraction of resting CD4 positive memory T cells harbor transcriptionally silent HIV provirus that can serve as a source of reemergent virus after cessation of highly active antiretroviral therapy. The mechanisms leading to transcriptional silencing and proviral latency are incopletely understood. Previous study identified the bromodomain-containing protein 4 (Brd4) gene as a hot spot for HIV integration specifically in latent populations, suggesting that this gene may be involved in HIV transcription and perhaps retroviral integration disrupts its fuction, leading to HIV latency. Brd4 is an important cellular target of viruses and appears to be intimately involved in the life cycle of various viruses, mainly through the interaction of Brd4 C-terminal domain and viral encoding proteins. We previously employed GST pull down and mass spectrum assay to discover a novel Brd4 C-terminal domain associated protein named as TAR DNA binding protein (TDP-43). TDP-43 functions in repressing HIV-1 transcription by binding to TAR DNA region of HIV-1 long terminal repeat. We therefore speculate that Brd4 might regulate HIV-1 transcription via the assciation with TDP-43. Since Brd4 tightly associates with interphase and mitotic chromsomes through its N-terminal bromodomains, we suppose that Brd4 may play a role in recruitment of TDP-43 to the chromsomes, leading to binding of TDP-43 to TAR DNA and repression of HIV-1 transcription.The current proposal is focused on the new mechanism of Brd4 on regulation of HIV-1 transcription. Firstly, Western blot, immunofluoresence, co-immunoprecipitation, Mammalian protein-protein interaction trap, and Bimolecular fluorescence complementation assay will be employed for further identification the interaction between Brd4 and TDP-43. Secondly, we will next investigate the effect of Brd4 on HIV-1 transcription by comparison the luciferase acitivity of HIV-1 LTR reporter when Brd4 is overexpressed or knocked down. Then .chromatin immunoprecipitation assay will be applied to dermine that Brd4 mediates repression of HIV-1 transcription via association with TDP-43. Finally, by construction of Brd4 N terminal truncation and Brd4 domain inhibitors, we will comfirm the association of Brd4 with chromosomes is necessary for its role in regulating HIV-1 transcription. We hope the study will provide an insight to regulatory mechanism of HIV-1 transcription and will contribute to a better understanding of the lantency mechsnism of HIV-1 in host cells, with ultimate goal to benefit the prevention and antiviral therapy of AIDS.

我们在前期工作中发现一个新的Brd4结合蛋白TDP-43，该蛋白可与HIV-1 TAR DNA结合抑制HIV-1转录。本项目拟研究Brd4通过与TDP-43结合调节HIV-1转录的新机制。首先通过蛋白印迹法、免疫荧光染色、免疫共沉淀、哺乳动物细胞蛋白-蛋白相互作用陷阱及双分子荧光互补技术，从细胞外、细胞内乃至活细胞水平验证Brd4与TDP-43之间的相互作用，明确TDP-43是一种新的Brd4结合蛋白；进一步通过荧光素酶实验研究Brd4过表达或基因沉默对HIV-1转录的影响；再通过染色体免疫共沉淀，明确Brd4通过TDP-43对HIV-1转录进行调节；最后构建Brd4 N端截短变构体或结构域抑制剂使Brd4从染色体上解离，研究Brd4与染色体结合对HIV-1转录的作用，探讨Brd4通过与TDP-43结合调节HIV-1转录的新机制。该研究将为进一步揭示HIV-1转录调控及潜伏机制提供新思路。

艾滋病是严重威胁人类健康的疾病，虽然近年来针对艾滋病的治疗已经取得显著性突破，尤其是通过抗逆转录病毒药物的联用，可有效清除患者体内HIV病毒的复制性感染；但HIV病毒感染CD4+T细胞后，有一部分病毒基因组会整合到宿主细胞的染色体上形成前病毒，以转录沉默的形式长期潜伏，一旦停止HAAT（highly active antiviral therapy，HAAT）治疗，这些前病毒就会重新开始复制成为复发感染源。目前对于导致HIV病毒转录沉默及前病毒潜伏性整合的机制还不清楚，研究HIV病毒在体内的转录调控及潜伏机制对艾滋病的防治具有重大意义。艾滋病病原体人类免疫缺陷病毒（HIV-1）的基因表达受到病毒因素和宿主因素的双重调控。TDP-43是一种转录抑制因子，可通过与整合到细胞染色体上的HIV-1前病毒基因组TAR DNA结合，抑制HIV-1的转录。另一种宿主蛋白Brd4能通过与PTEF-b结合，竞争性抑制Tat蛋白与PTEF-b结合，从而抑制HIV-1的转录。我们在前期工作中发现TDP-43有可能是一种新的Brd4结合蛋白，因此本项目拟研究Brd4通过与TDP-43结合抑制HIV-1转录的新机制。我们首先通过蛋白印迹法、免疫荧光染色、免疫共沉淀、哺乳动物细胞蛋白-蛋白相互作用陷阱等实验，从细胞外、细胞内乃至活细胞水平验证Brd4与TDP-43之间的相互作用，明确了TDP-43是一种新的Brd4结合蛋白；进一步通过荧光素酶实验证实Brd4可以负调控HIV-1的转录，当过表达Brd4时可显著抑制HIV-1启动子区的激活；而将Brd4敲除后可增强HIV-1启动子区的活性；最后通过染色体免疫共沉淀，证明将Brd4过表达时可增强TDP-43与HIV-LTR TAR DNA区域的结合；而将Brd4敲除时TDP-43与HIV-LTR TAR DNA区域的结合会受到明显抑制；并且，当用Brd4抑制剂JQ1处理细胞，使Brd4从染色体上解离时可显著抑制TDP-43与HIV-LTR TAR DNA区域的结合，证明Brd4与染色体结合对抑制HIV-1转录的作用是必须的。通过以上研究，证明Brd4通过其N端两个溴结构域与乙酰化染色体结合，并通过其C端与TDP-43结合并将TDP-43招募至HIV-1长末端重复序列的启动子区，发挥抑制HIV-1的转录的作用。