CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.
CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.
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DOI：
10.1371/journal.ppat.1003880
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发表时间：
2014-01
期刊：
影响因子：
6.7
通讯作者：
Swaminathan S
中科院分区：
文献类型：
--
作者：
Li DJ;Verma D;Mosbruger T;Swaminathan S
Kaposi肉瘤相关疱疹病毒(KSHV)是一种引起Kaposi肉瘤的人类疱疹病毒，与淋巴增生性疾病的发展有关。KSHV从潜伏期和病毒粒子的产生中重新激活依赖于80多个裂解循环基因的有效转录和病毒DNA复制。CTCF和粘附素是协同调节基因表达和介导远程DNA相互作用的细胞蛋白，已被证明与疱疹病毒基因组中的特定位置结合。CTCF和粘附素在潜伏期调节KSHV基因的表达，也可能控制裂解基因的重新激活，尽管它们在裂解基因表达中的作用尚不完全清楚。在这里，我们通过高分辨率染色质免疫沉淀和深度测序(ChIP-Seq)分析了KSHV病毒复活和病毒粒子产生过程中CTCF和粘附素结合的动态变化，并表明这两种蛋白都以不同的运动学和空间模式从病毒基因组中解离。通过利用siRNAs特异性地耗尽CTCF和粘附素成分Rad21，我们证明了这两种蛋白都是KSHV复制的有效限制因素，粘附素被击倒导致病毒产量增加数百倍。用高通量RNA测序分析了CTCF和粘附素耗竭对KSHV裂解转录的影响，结果表明这两种蛋白对KSHV裂解转录都有复杂的整体影响。具体地说，这两种蛋白最初都是病毒转录的积极因子，但后来抑制了KSHV的裂解转录，因此它们的净作用是限制KSHV RNA的积累。粘附素是一种比CTCF更有效的抑制KSHV转录的蛋白，但这两种蛋白也是有效转录KSHV基因子集所必需的。这些数据揭示了CTCF和粘附素在相对较小的基因组中转录的新效应，这些效应类似于它们作为某些启动子的基因特异性激活剂对细胞基因组的影响，但不同之处在于它们作为转录的全局负调控因子。卡波西肉瘤相关疱疹病毒(KSHV)是一种引起卡波西肉瘤和淋巴瘤的人类病毒。KSHV在B淋巴细胞中建立终身感染，并以环状DNA分子的潜伏形式持续存在。重新激活和复制产生具有感染性的病毒粒子，使潜伏感染得以传播和维持。控制再激活的细胞机制仍未完全确定。调节RNA转录的宿主蛋白在控制病毒重新激活方面发挥着重要作用。在这项研究中，我们使用高通量技术分析了当病毒重新激活产生感染性病毒粒子时，两种细胞蛋白CTCF和Rad21与KSHV基因组的结合。我们发现，当重新激活时，这些蛋白质会从潜在的基因组中解离。我们还发现，耗尽这些蛋白质的细胞会使病毒产量增加100倍。耗尽CTCF或Rad21细胞后，KSHV的RNA合成发生了复杂的变化，大多数KSHV RNA的数量大大增加。我们还表明，RAD21和CTCF是病毒有效合成RNA所必需的。我们的研究为细胞如何使用CTCF和Rad21来限制KSHV合成RNA和从潜伏期重新激活以产生传染性病毒的能力提供了新的见解。
Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus that causes Kaposi's sarcoma and is associated with the development of lymphoproliferative diseases. KSHV reactivation from latency and virion production is dependent on efficient transcription of over eighty lytic cycle genes and viral DNA replication. CTCF and cohesin, cellular proteins that cooperatively regulate gene expression and mediate long-range DNA interactions, have been shown to bind at specific sites in herpesvirus genomes. CTCF and cohesin regulate KSHV gene expression during latency and may also control lytic reactivation, although their role in lytic gene expression remains incompletely characterized. Here, we analyze the dynamic changes in CTCF and cohesin binding that occur during the process of KSHV viral reactivation and virion production by high resolution chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and show that both proteins dissociate from viral genomes in kinetically and spatially distinct patterns. By utilizing siRNAs to specifically deplete CTCF and Rad21, a cohesin component, we demonstrate that both proteins are potent restriction factors for KSHV replication, with cohesin knockdown leading to hundred-fold increases in viral yield. High-throughput RNA sequencing was used to characterize the transcriptional effects of CTCF and cohesin depletion, and demonstrated that both proteins have complex and global effects on KSHV lytic transcription. Specifically, both proteins act as positive factors for viral transcription initially but subsequently inhibit KSHV lytic transcription, such that their net effect is to limit KSHV RNA accumulation. Cohesin is a more potent inhibitor of KSHV transcription than CTCF but both proteins are also required for efficient transcription of a subset of KSHV genes. These data reveal novel effects of CTCF and cohesin on transcription from a relatively small genome that resemble their effects on the cellular genome by acting as gene-specific activators of some promoters, but differ in acting as global negative regulators of transcription. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human virus that causes Kaposi's sarcoma and lymphoma. KSHV establishes a lifelong infection in B lymphocytes, and persists in a latent form as circular DNA molecules. Reactivation and replication yield infectious virions, allowing transmission and maintenance of latent infection. The cellular mechanisms controlling reactivation remain incompletely characterized. Host proteins that regulate RNA transcription play an important role in controlling viral reactivation. In this study, we used high-throughput techniques to analyze the binding of two cellular proteins, CTCF and Rad21, to the KSHV genome as the virus reactivated to produce infectious virions. We found that these proteins dissociate from the latent genome when reactivation occurs. We also found that depleting cells of these proteins increases virus production as much as a hundredfold. Depleting the cell of CTCF or Rad21 caused complex changes in the synthesis of RNAs by KSHV, with the amounts of most KSHV RNAs increasing greatly. We also showed that Rad21 and CTCF are needed for the virus to synthesize RNAs efficiently. Our study provides new insights into how the cell uses CTCF and Rad21 to limit KSHV's ability to synthesize RNA and reactivate from latency to produce infectious virus.
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