ATRX limits the accessibility of histone H3-occupied HSV genomes during lytic infection.
ATRX limits the accessibility of histone H3-occupied HSV genomes during lytic infection.
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DOI：
10.1371/journal.ppat.1009567
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发表时间：
2021-04
期刊：
影响因子：
6.7
通讯作者：
Knipe DM
中科院分区：
文献类型：
--
作者：
Cabral JM;Cushman CH;Sodroski CN;Knipe DM
组蛋白在进入人成纤维细胞的细胞核后迅速加载到HSV基因组上，但组蛋白加载对病毒复制的影响尚未完全确定。我们最近发现，ATRX是从头沉积H3的HSV基因组后，核进入，但通过维持病毒异染色质限制感染。为了进一步研究ATRX和其他组蛋白H3分子伴侣在限制HSV中的作用，我们感染了系统性耗尽核H3分子伴侣的人成纤维细胞。我们发现ATRX/DAXX复合物在核H3分子伴侣中是独特的，其限制ICP0无效HSV感染的能力。只有ATRX的耗竭显著减轻了病毒复制的限制。有趣的是，没有单独的核H3分子伴侣需要沉积到输入病毒基因组上，这表明在裂解感染期间，H3沉积可能通过多种途径发生。在对照和用ICP0无效HSV感染的ATRX-KO细胞中的总组蛋白H3的ChIP-seq显示HSV DNA在整个病毒基因组中负载有高水平的组蛋白。尽管存在高水平的H3，但ATAC-seq分析显示HSV DNA是高度可接近的，特别是在GC含量高的区域，并且在裂解感染期间大部分没有组织成有序的核小体。ATRX降低了病毒DNA对TN5转座酶活性的可及性，并增强了与核小体样结构相关的病毒DNA片段大小的积累。总之，这些发现支持了一个模型，其中ATRX通过改变组蛋白H3加载的病毒染色质的结构来限制病毒感染，从而降低病毒DNA转录的可及性。HSV基因组的高GC富集区域，特别是HSV-1基因组的S组分反向重复序列，显示出增加的可接近性，这可能导致在感染起始期间转录这些区域中编码的IE基因的能力增加。单纯疱疹病毒（HSV）的基因组受宿主细胞过程的影响，这些过程将DNA组织成有序的结构，称为染色质，并起到调节基因表达的作用。在进入宿主细胞核后不久，HSV DNA与组蛋白结合，组蛋白是DNA包裹的核小体的蛋白质亚基。我们评估了已知的组蛋白调节蛋白将组蛋白装载到HSV DNA上的能力。只有这些因子的组合耗竭才能降低病毒DNA上的组蛋白水平，揭示了多个组蛋白伴侣途径促进HSV DNA上的染色质负载。然而，组蛋白装载剂之一，ATRX，是显着更有效地限制HSV复制比其他因素。虽然ATRX因其在维持细胞DNA稳定性中的作用而广为人知，但它也被称为DNA病毒的一般限制因子。为了研究ATRX限制HSV的独特机制，我们使用基于高分辨率测序的测定来评估HSV DNA的可及性。我们发现ATRX在感染过程中降低了病毒DNA的可及性，可能阻止了病毒基因表达和复制所必需的因子的获得。我们的研究提供了新的见解ATRX作为一种广义的DNA病毒限制因子的机制。
Histones are rapidly loaded on the HSV genome upon entry into the nucleus of human fibroblasts, but the effects of histone loading on viral replication have not been fully defined. We showed recently that ATRX is dispensable for de novo deposition of H3 to HSV genomes after nuclear entry but restricted infection through maintenance of viral heterochromatin. To further investigate the roles that ATRX and other histone H3 chaperones play in restriction of HSV, we infected human fibroblasts that were systematically depleted of nuclear H3 chaperones. We found that the ATRX/DAXX complex is unique among nuclear H3 chaperones in its capacity to restrict ICP0-null HSV infection. Only depletion of ATRX significantly alleviated restriction of viral replication. Interestingly, no individual nuclear H3 chaperone was required for deposition of H3 onto input viral genomes, suggesting that during lytic infection, H3 deposition may occur through multiple pathways. ChIP-seq for total histone H3 in control and ATRX-KO cells infected with ICP0-null HSV showed that HSV DNA is loaded with high levels of histones across the entire viral genome. Despite high levels of H3, ATAC-seq analysis revealed that HSV DNA is highly accessible, especially in regions of high GC content, and is not organized largely into ordered nucleosomes during lytic infection. ATRX reduced accessibility of viral DNA to the activity of a TN5 transposase and enhanced accumulation of viral DNA fragment sizes associated with nucleosome-like structures. Together, these findings support a model in which ATRX restricts viral infection by altering the structure of histone H3-loaded viral chromatin that reduces viral DNA accessibility for transcription. High GC rich regions of the HSV genome, especially the S component inverted repeats of the HSV-1 genome, show increased accessibility, which may lead to increased ability to transcribe the IE genes encoded in these regions during initiation of infection. The genome of herpes simplex virus (HSV) is subject to host cell processes that organize DNA into ordered structures, known as chromatin, and function to regulate gene expression. Shortly after entry to the host cell nucleus, HSV DNA becomes associated with histones, protein subunits of nucleosomes around which DNA is wrapped. We evaluated the ability of known histone regulatory proteins to load histones onto HSV DNA. Only combinatorial depletion of these factors could reduce the levels of histones on viral DNA, revealing that multiple histone chaperone pathways promote chromatin loading on HSV DNA. However, one of the histone-loaders, ATRX, was significantly more potent in restricting HSV replication than the other factors. While ATRX is widely known for its role in maintaining cellular DNA stability, it is also known to act as a general restriction factor for DNA viruses. To investigate the unique mechanism by which ATRX restricts HSV, we used a high-resolution sequencing-based assay to assess the accessibility of HSV DNA. We found that ATRX promotes reduced accessibility of viral DNA during infection, likely preventing access for factors necessary for viral gene expression and replication. Our study provides new insight into the mechanism by which ATRX acts as a generalized DNA virus restriction factor.
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