中国科学院昆明动物研究所机构知识库

I型单纯疱疹病毒是一类线性双链DNA病毒，是口唇疱疹和病毒性角膜炎的主要诱发因素，在人群中有着广泛的潜伏感染。I型单纯疱疹病毒在体外感染细胞后进入裂解感染，迅速控制宿主转录机器、细胞周期和细胞凋亡等过程，为病毒基因的转录和复制创造条件。在这一过程中，许多细胞和病毒因子起到关键的调控作用。CTCF是真核细胞中广泛表达的一种锌指蛋白，具有组织染色质高级结构、调控基因转录、作为绝缘子蛋白、参与遗传印记和参与选择性剪接等多种功能。AGO2蛋白是小RNA介导的基因沉默过程的关键因子，在真核生物中广泛存在。目前认为AGO2的主要功能是通过结合小RNA实现对mRNA的切割、转录后抑制等调控基因表达的作用，这些过程多发生在细胞质中。但随着研究的深入，越来越多的证据表明AGO2能够被转运到细胞核中，参与一些核内生物过程，如选择性剪切、DNA损伤修复等。已有报道CTCF在I型单纯疱疹病毒潜伏期可以结合在病毒基因组上并且帮助维持潜伏期感染。但CTCF在I型单纯疱疹病毒裂解感染中的功能和作用机制还不清楚。目前还没有关于AGO2蛋白在HSV-1感染过程中作用的研究。在本论文中我们利用免疫荧光实验发现CTCF能够被HSV-1招募到病毒的转录复制区，AGO2能够被HSV-1诱导上调表达并入核。免疫印记实验发现CTCF在病毒感染过程中未受影响，而AGO2表现为核内部分增加一条约130 kDa的条带。利用染色质免疫共沉淀结合高通量测序（ChIP-seq）的方法，我们发现CTCF在I型单纯疱疹病毒裂解感染的早期即可结合到病毒基因组上，并且结合位点与潜伏期结合位点不同。ChIP-seq实验表明CTCF在裂解期I型单纯疱疹病毒基因组上有25个结合位点，广泛分布于病毒基因，基因间和调控区域。敲低CTCF后在不影响细胞状态的情况下，会导致病毒转录，复制和病毒颗粒产量下降。而AGO2缺失并不对HSV-1的转录和复制造成直接广泛的影响，可能特异性的对个别基因进行调控（比如UL36和UL44），但是总体上对病毒增殖有两面性的作用，在病毒感染初期，AGO2缺失影响病毒增殖，随着感染时间延长，AGO2缺失有利于病毒增殖。进一步的实验表明CTCF通过阻止异染色质蛋白H3K9me3的方式给予病毒基因组开放的状态。CTCF还可以通过促进RNA聚合酶II（RNA Pol II）结合病毒基因组的方式促进病毒转录。CTCF敲低后，代表活跃转录的RNA Pol II磷酸化形式RNA Pol II ser2在病毒基因上的结合减少，而代表转录停顿的RNA Pol II磷酸化形式RNA Pol II ser5更多的结合到病毒基因上。这些结果证明CTCF可以通过阻止病毒基因组异染色质化，同时促进转录活跃型RNA Pol II结合到病毒基因组上来促进病毒转录。

Herpes Simplex Virus type 1 (HSV-1) is a ubiquitous and important human pathogen, with linear and double-stranded DNA genome and is responsible for herpes labialis and viral keratitis. HSV-1 enters productive or lytic infection in many cell types, where it will take control of host cellular transcription machinery, cell cycle and apoptosis regulation to facilitate viral transcription and replication. Many cellular and viral factors participate in this process. CTCF is a zinc finger protein with functions in nuclear organization, transcription regulation, chromatin insulation, imprinting, alternative splicing and other important nuclear processes. AGO2 is an important factor in micro RNA induced RNA silencing processing. Recent evidence suggest that AGO2 can translocate into nucleus and participates in DNA damage repair, alternative splicing and transcriptional silencing and activation. It has reported that CTCF interacts with the latent HSV-1 genome and may control its latency by protecting an area of active chromatin around the LAT promoter from silencing. However, whether CTCF plays a role in HSV-1 lytic infection is not known. In contrast, whether AGO2 has a role in HSV-1 lytic infection has not been analyzed. In this study, we analyzed the functions of both CTCF and AGO2 in HSV-1 lytic infection. Here, we report that CTCF is recruited to HSV-1 replication compartments. AGO2 is induced increased expression and translocate into nucleus. There was an additional band of AGO2 at 130 kDa determined by Western Blot after HSV-1 infection, which may be the modified form of AGO2 locating in the nucleus. While there is a little effect on the amount and molecular weight of CTCF protein. Using Chromatin Immunoprecipitation combined with high-hroughput sequencing (ChIP - seq), we find that CTCF interacts extensively with HSV-1 genome though 25 bingding sites, which are totally different from latency infection. Knocking down CTCF disrupted these structures and resulted in reduction of viral protein levels, viral gene transcription, viral genome copy number and viral yield. While deletion of AGO2 had no global effects on viral protein level but UL36 and UL44, indicating that AGO2 may plays specific roles in viral gene regulation, or RNA processing. AGO2 deletion had a duel effect on viral production. At the early stage of infection, deletion of AGO2 prevented viral proliferation, while at the late stage of infection, AGO2 deficiency will augment viral production. On viral chromatin, the knockdown of CTCF led to an increase of H3K9me3 and a reduction of RNA pol II recruitment to viral genes. Importantly, ChIP-seq revealed that there is a higher level of Ser2P vs Ser5P forms of RNA Pol II near CTCF binding sites in the viral genome, and CTCF knockdown resulted in a reduction of Ser2P but an increase of Ser5P forms of RNA Pol II on viral genes. These results suggest that CTCF promotes HSV-1 lytic transcription by facilitating binding and elongation of RNA Pol II, while preventing silenced chromatin on the viral genome.