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

项目概要/摘要： 单纯疱疹病毒1型（HSV-1）感染全世界估计有37亿50岁以下的人。 感染的首要症状是口唇病变，但临床上严重的疾病如眼部角膜炎 和脑炎，特别是在免疫功能低下的个体中。在溶解性和非溶解性之间交替的能力 上皮细胞中的复制和神经元中的大量休眠潜伏期是HSV-1广泛流行的基础。 为了复制和传播，HSV-1必须抵消和吸收宿主细胞的表观遗传机制。虽然 病毒粒子内的病毒DNA不与组蛋白结合，HSV-1 DNA快速组装成核小体 在进入宿主细胞核时。为了使生产性裂解复制发生，病毒蛋白质必须能够使过渡 病毒染色质从最初的抑制性染色质状态转变为更宽松的非凝聚状态。这 该提案将研究一种新的机制，HSV-1通过这种机制引导病毒染色质的重组。 虽然已经鉴定了具有前病毒功能的染色质修饰酶和组蛋白修饰酶， 几乎没有与增强的病毒产生相关的组蛋白变体。我们最近发现了 组蛋白H2A.Bbd作为一种新的HSV-1宿主因子，H2A.Bbd缺失降低野生型HSV-1产量。 将这种组蛋白变体整合到细胞核小体中与两种活跃转录的基因有关 和允许的染色质状态。H2A.Bbd也靶向宿主DNA合成位点并在其上富集。 我们的初步研究表明H2A.Bbd在HSV-1裂解复制中起作用，以增强病毒DNA合成。 此外，H2A.Bbd组蛋白伴侣NAP 1 L1先前被鉴定为与HSV-1相关， DNA复制蛋白ICP 8。已建立的H2A.Bbd掺入与解聚 染色质表明，其掺入HSV-1染色质可能增加病毒DNA的可及性， 增强HSV-1 DNA接近病毒复制蛋白。我们假设组蛋白变体 H2A.Bbd通过NAP 1 L1介导的掺入促进裂解复制期间的病毒DNA合成 HSV-1相关核小体以建立容许的染色质状态。该提案旨在解决 H2A.Bbd是否被募集到复制灶以促进病毒DNA合成（目的1），以及H2A.Bbd是否被 通过NAP 1 L1掺入HSV-1相关核小体，改变病毒染色质并促进病毒DNA 无障碍环境（目标2）。目的1将测试通过以下方法是否降低H2A.Bbd耗尽细胞中的总病毒DNA合成水平： qPCR（目标1.1）;通过显微镜检查研究H2A.Bbd向复制灶的募集（目标1.2）;并比较 通过iPOND-MS在存在和不存在H2A.Bbd的情况下复制蛋白与病毒DNA的缔合 (Aim 1.3）。目标2将确定H2A.Bbd是否通过ChIP掺入病毒染色质（目标2.1），并检查 H2A.Bbd对ATAC-Seq病毒DNA可及性的影响（目的2.2）。拟议项目旨在提供 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性操作抑制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.