Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis

Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact contribution is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML NBs, leading to the formation of viral DNA-containing PML NBs (vDCP NBs), and the complete silencing of HSV-1. Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP and Re-ChIP show that vDCP NBs-associated latent/quiescent viral genomes are chromatinized almost exclusively with H3.3 modified on its lysine (K) 9 by trimethylation, consistent with an interaction of the H3.3 chaperones with multiple viral loci and with the transcriptional silencing of HSV-1. Only simultaneous inactivation of both H3.3 chaperone complexes has a significant impact on the deposition of H3.3 on viral genomes, suggesting a compensation mechanism. In contrast, the sole depletion of PML significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. vDCP NBs-associated HSV-1 genomes are not definitively silenced since the destabilization of vDCP NBs by ICP0, which is essential for HSV-1 reactivation in vivo, allows the recovery of a transcriptional lytic program and the replication of viral genomes. Consequently, the present study demonstrates a specific chromatin regulation of vDCP NBs-associated latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML NB/histone H3.3/H3.3 chaperone axis.

单纯疱疹病毒1型（Herpes simplex virus 1, HSV-1）的潜伏建立过程受早幼粒细胞白血病核体（promyelocytic leukemia nuclear bodies, PML NBs，又称ND10）的严格调控，但其确切作用机制仍不明晰。HSV-1潜伏感染的标志性特征是潜伏病毒基因组与PML NBs发生相互作用，进而形成含病毒DNA的PML NBs（vDCP NBs），并实现HSV-1的完全转录沉默。本研究采用可重现vDCP NBs形成的复制缺陷型HSV-1感染人原代成纤维细胞模型，结合用于检测潜伏/静止期HSV-1的免疫荧光原位杂交（immuno-FISH）技术，证实vDCP NBs中同时存在组蛋白H3.3（histone H3.3）及其组蛋白伴侣复合物——DAXX/ATRX与HIRA复合物（HIRA、UBN1、CABIN1及ASF1a）。HIRA同样可与HSV-1感染的野生型小鼠三叉神经节（trigeminal ganglia, TG）神经元内的vDCP NBs发生共定位。染色质免疫沉淀（ChIP）及重染色质免疫沉淀（Re-ChIP）实验结果显示，与vDCP NBs结合的潜伏/静止期病毒基因组几乎完全以经赖氨酸（K）9三甲基化修饰的H3.3形式完成染色质组装，这一结果与H3.3伴侣复合物与多个病毒基因座的相互作用及HSV-1的转录沉默状态相一致。仅同时灭活两种H3.3伴侣复合物时，病毒基因组上的H3.3沉积才会受到显著影响，提示存在代偿机制。与之相反，仅敲除PML会显著干扰潜伏/静止期病毒基因组与H3.3的染色质组装，且未出现整体替换为H3.1的情况。vDCP NBs结合的HSV-1基因组并非被永久沉默：体内HSV-1复激活所必需的感染细胞蛋白0（ICP0）可破坏vDCP NBs，该过程可恢复病毒的裂解性转录程序并实现病毒基因组复制。综上，本研究证实，通过涉及两种H3.3伴侣复合物DAXX/ATRX与HIRA的H3.3依赖性途径，vDCP NBs结合的潜伏/静止期HSV-1基因组受到特异性染色质调控。此外，本研究还揭示，PML NBs通过PML NBs/组蛋白H3.3/H3.3伴侣轴，在潜伏/静止期HSV-1的H3.3染色质组装中发挥核心调控作用。