Incorporation of a histone variant into viral chromatin to promote herpes simplex virus replication

项目摘要/摘要： 据估计，全世界有37亿50岁以下的人感染单纯疱疹病毒1型(HSV-1)。 感染的主要症状是口腔损害，但临床上严重的疾病，如眼角膜炎 而脑炎可能会导致，特别是在免疫功能低下的人身上。在裂解之间交替的能力 上皮细胞的复制和神经元的大量休眠潜伏期是单纯疱疹病毒1型S广泛流行的基础。 为了复制和传播，HSV-1必须同时抵消和选择宿主细胞的表观遗传机制。虽然 病毒粒子内的病毒DNA与组蛋白无关，HSV-1 DNA迅速组装成核小体 在进入宿主核时。为了发生有效的裂解复制，病毒蛋白必须使这种转变成为可能。 病毒染色质从最初的抑制染色质状态转变为更允许的去浓缩状态。这 提案将研究一种新的机制，HSV-1通过它导航病毒染色质的这种重组。 尽管已经鉴定出具有亲病毒功能的染色质修饰酶和组蛋白修饰酶， 很少有组蛋白变体与增强病毒产生有关。我们最近发现了这种变种 组蛋白H2A.bbd是一种新的HSV-1宿主因子，其缺失会降低野生型HSV-1的产量。 将这种组蛋白变体结合到细胞核小体中与两个活跃转录的基因相关 和一种允许的染色质状态。H2A.Bbd也以宿主DNA合成为靶点并在其上富含。 我们的初步研究表明，H2A.Bbd在HSV-1裂解复制中起作用，以促进病毒DNA的合成。 此外，Hbd组蛋白伴侣NAP1L1与HSV-1相关 DNA复制蛋白ICP8。H_2A.BBD掺入与去缩合的已建立的联系 染色质表明，它与HSV-1染色质的结合可能会增加病毒DNA的可及性，潜在地 增强单纯疱疹病毒1型DNA对病毒复制蛋白的访问。我们假设组蛋白变体 H2A.Bbd通过NAP1L1介导的掺入促进裂解复制过程中病毒DNA的合成 与HSV-1相关的核小体建立一种允许的染色质状态。这项提案旨在解决 是否将H2A.bbd招募到复制中心以促进病毒DNA合成(目标1)以及是否 NAP1L1进入HSV-1相关核小体改变病毒染色质和促进病毒DNA 无障碍(目标2)。AIM 1将测试通过以下方法是否降低了H2A.Bbd耗尽细胞的总病毒DNA合成水平 定量聚合酶链式反应(目标1.1)；通过显微镜研究H2A.Bbd向复制灶的募集(目标1.2)；并比较 IPOND-MS检测有无H_2A.Bbd时复制蛋白与病毒DNA的结合 (目标1.3)。Aim 2将通过芯片(Aim 2.1)确定是否将H2A.bbd结合到病毒染色质中，并检查 过氧化氢对ATAC-Seq获得病毒DNA的影响(目标2.2)。拟议的项目旨在提供 对病毒DNA合成的表观遗传机制的新见解，同时可能揭示新的 通过病毒染色质的治疗操作抑制HSV-1和相关疱疹病毒的策略。

Project Summary/Abstract: Herpes simplex virus 1 (HSV-1) infects an estimated 3.7 billion people under the age of 50 worldwide. The primary symptoms of infection are orolabial lesions, but clinically severe diseases such as ocular keratitis and encephalitis can result, particularly in immunocompromised individuals. The ability to alternate between lytic replication in the epithelia and a largely dormant latency in neurons underpins HSV-1’s wide-spread prevalence. To replicate and spread, HSV-1 must both counteract and co-opt the host cell epigenetic machinery. Although viral DNA within the virion is not associated with histones, HSV-1 DNA is rapidly assembled into nucleosomes upon entry into the host nucleus. For productive lytic replication to occur, viral proteins must enable the transition of viral chromatin from this initial repressive chromatin state to a more permissive, de-condensed state. This proposal will investigate a novel mechanism by which HSV-1 navigates this reorganization of viral chromatin. Although chromatin-modifying and histone-modifying enzymes with pro-viral functions have been identified, there have been few histone variants linked to enhanced virus production. We recently identified the variant histone H2A.Bbd as a novel HSV-1 host factor, with H2A.Bbd depletion reducing wild-type HSV-1 yields. Incorporation of this histone variant into cellular nucleosomes is associated with both actively transcribed genes and a permissive chromatin state. H2A.Bbd is also targeted to and enriched at sites of host DNA synthesis. Our initial studies suggest that H2A.Bbd acts in HSV-1 lytic replication to enhance viral DNA synthesis. Furthermore, the H2A.Bbd histone chaperone NAP1L1 was identified previously as associating with the HSV-1 DNA replication protein ICP8. The established association of H2A.Bbd incorporation with de-condensed chromatin suggests that its incorporation into HSV-1 chromatin may increase viral DNA accessibility, potentially enhancing the access of HSV-1 DNA to viral replication proteins. We hypothesize that the histone variant H2A.Bbd promotes viral DNA synthesis during lytic replication by NAP1L1-mediated incorporation into HSV-1-associated nucleosomes to establish a permissive chromatin state. This proposal seeks to address whether H2A.Bbd is recruited to replication foci to promote viral DNA synthesis (Aim 1) and whether H2A.Bbd is incorporated into HSV-1-associated nucleosomes by NAP1L1 to alter viral chromatin and promote viral DNA accessibility (Aim 2). Aim 1 will test if total viral DNA synthesis levels are reduced in H2A.Bbd-depleted cells by qPCR (Aim 1.1); investigate recruitment of H2A.Bbd to replication foci by microscopy (Aim 1.2); and compare the association of replication proteins with viral DNA in the presence and absence of H2A.Bbd by iPOND-MS (Aim 1.3). Aim 2 will determine if H2A.Bbd is incorporated into viral chromatin by ChIP (Aim 2.1) and examine the impact of H2A.Bbd on viral DNA accessibility by ATAC-Seq (Aim 2.2). The proposed project aims to provide new insights into the epigenetic mechanisms underlying viral DNA synthesis, while potentially revealing new strategies for inhibiting HSV-1 and related herpes viruses via the therapeutic manipulation of viral chromatin.