Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA.
Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA.
J Virol. 2019 Jun 14;93(13). doi: 10.1128/JVI.00415-19. Print 2019 Jul 1.
The cellular insulator protein CTCF plays a role in herpes simplex virus 1 (HSV-1) latency through the establishment and regulation of chromatin boundaries. We previously found that the CTRL2 regulatory element downstream from the latency-associated transcript (LAT) enhancer was bound by CTCF during latency and underwent CTCF eviction at early times postreactivation in mice latently infected with 17+ virus. We also showed that CTRL2 was a functional enhancer-blocking insulator in both epithelial and neuronal cell lines. We hypothesized that CTRL2 played a direct role in silencing lytic gene expression during the establishment of HSV-1 latency. To test this hypothesis, we used a recombinant virus with a 135-bp deletion spanning only the core CTRL2 insulator domain (ΔCTRL2) in the 17+ background. Deletion of CTRL2 resulted in restricted viral replication in epithelial cells but not neuronal cells. Following ocular infection, mouse survival decreased in the ΔCTRL2-infected cohort, and we found a significant decrease in the number of viral genomes in mouse trigeminal ganglia (TG) infected with ΔCTRL2, indicating that the CTRL2 insulator was required for the efficient establishment of latency. Immediate early (IE) gene expression significantly increased in the number of ganglia infected with ΔCTRL2 by 31 days postinfection relative to the level with 17+ infection, indicating that deletion of the CTRL2 insulator disrupted the organization of chromatin domains during HSV-1 latency. Finally, chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) analyses of TG from ΔCTRL2-infected mice confirmed that the distribution of the repressive H3K27me3 (histone H3 trimethylated at K27) mark on the ΔCTRL2 recombinant genomes was altered compared to that of the wild type, indicating that the CTRL2 site modulates the repression of IE genes during latency. It is becoming increasingly clear that chromatin insulators play a key role in the transcriptional control of DNA viruses. The gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) utilize chromatin insulators to order protein recruitment and dictate the formation of three-dimensional DNA loops that spatially control transcription and latency. The contribution of chromatin insulators in alphaherpesvirus transcriptional control is less well understood. The work presented here begins to bridge that gap in knowledge by showing how one insulator site in HSV-1 modulates lytic gene transcription and heterochromatin deposition as the HSV-1 genome establishes latency.
细胞绝缘子蛋白 CTCF 在单纯疱疹病毒 1（HSV-1）潜伏期中发挥作用，通过建立和调节染色质边界。我们之前发现，潜伏相关转录物（LAT）增强子下游的 CTRL2 调节元件在潜伏期间与 CTCF 结合，并在潜伏感染 17+病毒的小鼠重新激活后早期发生 CTCF 驱逐。我们还表明，CTRL2 是上皮细胞和神经元细胞系中具有功能的增强子阻断绝缘子。我们假设 CTRL2 在 HSV-1 潜伏期建立过程中直接沉默裂解基因表达。为了验证这一假设，我们使用了一种重组病毒，该病毒在 17+背景下跨越仅核心 CTRL2 绝缘子域的 135 个碱基对缺失（ΔCTRL2）。删除 CTRL2 导致上皮细胞中的病毒复制受到限制，但在神经元细胞中没有。眼部感染后，感染 ΔCTRL2 的小鼠存活率降低，我们发现感染 ΔCTRL2 的小鼠三叉神经节（TG）中的病毒基因组数量显著减少，表明 CTRL2 绝缘子对于潜伏期的有效建立是必需的。与 17+感染相比，感染 ΔCTRL2 的神经节中立即早期（IE）基因表达数量显著增加，表明删除 CTRL2 绝缘子会破坏 HSV-1 潜伏期期间染色质域的组织。最后，对感染 ΔCTRL2 的小鼠 TG 的高通量测序（ChIP-seq）分析表明，与野生型相比，ΔCTRL2 重组基因组上抑制性 H3K27me3（组蛋白 H3 在 K27 处三甲基化）标记的分布发生改变，表明 CTRL2 位点在潜伏期内调节 IE 基因的抑制。越来越明显的是，染色质绝缘子在 DNA 病毒的转录控制中起着关键作用。γ疱疹病毒 EBV 和卡波西肉瘤相关疱疹病毒（KSHV）利用染色质绝缘子来排列蛋白质募集，并决定三维 DNA 环的形成，从而空间控制转录和潜伏。α疱疹病毒转录控制中染色质绝缘子的贡献了解较少。本文的工作通过展示 HSV-1 中的一个绝缘子位点如何调节裂解基因转录和异染色质沉积，开始填补这一知识空白，因为 HSV-1 基因组建立潜伏。
