Abstract：
Human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional transcription factor that is essential for lytic HCMV infection. IE2 functions as an activator of viral early genes, negatively regulates its own promoter, and is required for viral replication. The mechanisms by which IE2 executes these distinct functions are incompletely understood. Using PRO-Seq, which profiles nascent transcripts, and a recently developed DFF-chromatin immunoprecipitation (DFF-ChIP; employs chromatin digestion by the endonuclease DNA fragmentation factor prior to IP) approach that resolves occupancy and local chromatin environment, we show that IE2 controls viral gene transcription in three distinct capacities during late HCMV infection and reveal mechanisms that involve direct binding of IE2 to viral DNA. IE2 represses a subset of viral promoters by binding within their core promoter regions and blocking the assembly of preinitiation complexes (PICs). Remarkably, IE2 forms a repressive complex at the major immediate-early promoter region involving direct association of IE2 with nucleosomes and TBP. IE2 stimulates transcription by binding nearby, but not within, core promoter regions. In addition, IE2 functions as a direct roadblock to transcription elongation. At one locus, this function of IE2 appears to be important for the synthesis of a spliced viral RNA. Consistent with the minimal observed effects of IE2 depletion on host gene transcription, IE2 does not functionally engage the host genome. Our results reveal mechanisms of transcriptional control by IE2, uncover a previously unknown function of IE2 as a Pol II elongation modulator, and demonstrate that DFF-ChIP is a useful tool for probing transcription factor occupancy and interactions between transcription factors and nucleosomes at high resolution. IMPORTANCE HCMV infects more than half of the world population and persists lifelong in its hosts. Although generally asymptomatic, HCMV infection can lead to life-threating disease in immunosuppressed individuals. Moreover, HCMV is the leading infectious cause of birth defects in the United States. As there are no vaccines effective against HCMV and antiviral drugs exhibit toxicity and are undermined by resistant HCMV variants, other vulnerabilities in HCMV must be explored. Here, we characterize the mechanism by which IE2 controls transcription during late HCMV infection. We demonstrate that IE2 engages numerous consensus sites across the HCMV genome and functions as an activator, repressor, or elongation modulator depending on the context of IE2 binding sites in relation to Pol II initiation and elongation complexes. Our findings have important implications for the ongoing exploration of IE2 as an antiviral drug target.
摘要：
人巨细胞病毒（HCMV）立即早期2（IE2）蛋白是一种多功能转录因子，对于裂解性HCMV感染至关重要。IE2作为病毒早期基因的激活剂，负调节自己的启动子，并且是病毒复制所必需的。IE2执行这些不同功能的机制尚未完全理解。使用PRO-Seq，它描述了新生的成绩单，和最近开发的DFF-染色质免疫沉淀（DFF-ChIP；在IP之前通过核酸内切酶DNA片段化因子进行染色质消化）方法解决了占用和局部染色质环境，我们表明IE2在HCMV感染晚期以三种不同的能力控制病毒基因转录，并揭示了IE2与病毒DNA直接结合的机制。IE2通过在其核心启动子区域内结合并阻断预起始复合物(PIC)的组装来抑制病毒启动子的子集。值得注意的是,IE2在主要的立即早期启动子区域形成抑制复合物，涉及IE2与核小体和TBP的直接缔合。IE2通过附近的结合刺激转录，但不在内部，核心启动子区域。此外,IE2充当转录延伸的直接障碍。在一个地点，IE2的这种功能似乎对于剪接的病毒RNA的合成很重要。与观察到的IE2耗竭对宿主基因转录的最小影响一致，IE2在功能上不参与宿主基因组。我们的结果揭示了IE2的转录控制机制，揭示了IE2作为PolII延伸调节剂的先前未知的功能，并证明DFF-ChIP是高分辨率探测转录因子占有率以及转录因子与核小体之间相互作用的有用工具。重要性HCMV感染了世界一半以上的人口，并在其宿主中终生存在。虽然一般无症状，HCMV感染可导致免疫抑制个体的生命威胁疾病。此外，HCMV是美国出生缺陷的主要传染性原因。由于没有有效对抗HCMV的疫苗和抗病毒药物表现出毒性和被HCMV抗性变体破坏,必须探索HCMV中的其他漏洞。这里,我们描述了IE2在晚期HCMV感染期间控制转录的机制。我们证明了IE2在整个HCMV基因组中参与了许多共识位点，并充当激活剂，抑制子，或延伸调节剂，取决于IE2结合位点与PolII起始和延伸复合物相关的背景。我们的发现对IE2作为抗病毒药物靶标的持续探索具有重要意义。
