Molecular Genetics of HSV Reactivation

HSV 再激活的分子遗传学

• 批准号: 8318566
• 负责人: David C. Bloom
• 金额: $ 39.01万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8318566
• 关键词: Afferent Neurons; Antiviral Agents; Beryllium; Binding; Blindness; Cells; Chromatin; Chromosomes; Clinical; Complex; DNA; Data; Development; Disease; EZH2 gene; Elements; Encephalitis; Epigenetic Process; Episome; Event; Functional RNA; Ganglia; Gene Silencing; Genes; Genetic Transcription; Genome; Goals; Herpes Labialis; Herpesvirus 1; Heterochromatin; Histone H3; Human; Immunoprecipitation; Infection; Introns; Life; Lysine; Lytic; Maintenance; Malignant Neoplasms; Maps; Mediating; Modeling; Molecular Genetics; Mus; Neurons; PRC1 Protein; Play; Polycomb; Process; Proteins; RNA; Recruitment Activity; Recurrence; Regulation; Relative (related person); Repression; Research; Role; Simplexvirus; Small Interfering RNA; Specificity; Stimulus; Structure; Testing; Viral; Viral Genome; Virus Latency; Work; YY1 Transcription Factor; cell growth regulation; flexibility; inhibitor/antagonist; insight; knock-down; latent infection; lytic gene expression; mutant; novel; promoter; protein complex; research study; small molecule

DESCRIPTION (provided by applicant): Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection within sensory neurons. During latency the viral genomes are maintained as circular episomes and the lytic genes are silenced. Periodically the genomes within some of the neurons reactivate resulting in recurrent clinical disease. A major focus of our research is to determine how HSV-1 lytic genes are silenced in neurons during latency, and how this process is reversed during reactivation. During the past project period our studies have implicated the polycomb repressor complex (PRC) as an important epigenetic regulator of HSV latency and reactivation. The overall hypothesis to be tested in this proposal is that viral regulation of PRC binding to the HSV genome plays a key role in controlling the degree of suppression of lytic genes during latency in a manner that facilitates reactivation. The experiments proposed here will define the viral elements that are involved in this process. In Aim 1 we will identify the regions of the HSV-1 genome that recruit the polycomb establishment complex (PRC2). Cellular genes silenced by H3K27triMe recruit PRC2 via cis DNA elements or indirectly through interactions with YY1 or non-coding RNAs (ncRNAs). PRC2 then methylates histone H3 at lysine 27 which leads to heterochromatin formation. Therefore we will determine if PRC2 binds directly to elements on the HSV-1 genome, or whether this binding is mediated through other factors. After heterochromatin is established a second PRC, the PRC maintenance complex (PRC1), recognizes the H3K27triMe mark and maintains the repressed state. RNA immunoprecipitation (RIP) data indicate that the LAT 2.0kb stable intron binds to the PRC1 suggesting that the intron sequesters PRC1 and reduces the degree of H3K27triMe repression on the latent genomes. We hypothesize that this competition with PRC1 binding is a critical factor in maintaining the lytic genes in a more flexible "suppressed but reversible" heterochromatic state facilitating reactivation. In Aim 2 we will characterize the binding of PRC1 to the LAT intron and determine if reducing the amount of PRC1 in the cell is suficient to enhance HSV reactivation. Finally, in order for reactivation to occur, the PRC1 repression must be released and the chromatin associated with the HSV-1 lytic promoters must remodel to a transcriptionaly permissive state. We have previously shown that during explant-induced reactivation of latent murine ganglia that there is a transient increase in LAT abundance. The goal of Aim 3 will be to determine how the H3K27triMe mark is removed in order to allow lytic transcription to proceed, and whether the LAT is directly involved in this process. The proposed studies will provide key insight into the mechanism of lytic gene silencing during HSV-1 latency and details concerning the novel role that the LAT intron plays in modulating H3K27triMe. In addition this work may provide new insight into novel mechanisms of PRC regulation of cellular genes.

描述（由申请人提供）：1 型单纯疱疹病毒 (HSV-1) 在感觉神经元内建立终生潜伏感染。在潜伏期，病毒基因组保持为环状附加体，并且裂解基因被沉默。一些神经元内的基因组定期重新激活，导致临床疾病复发。我们研究的一个主要重点是确定 HSV-1 裂解基因在潜伏期如何在神经元中沉默，以及该过程如何在重新激活期间逆转。在过去的项目期间，我们的研究表明多梳阻遏复合物（PRC）是 HSV 潜伏期和重新激活的重要表观遗传调节因子。本提案要测试的总体假设是，PRC 与 HSV 基因组结合的病毒调节在以促进重新激活的方式控制潜伏期间裂解基因的抑制程度方面发挥着关键作用。这里提出的实验将定义参与该过程的病毒元素。在目标 1 中，我们将识别 HSV-1 基因组中招募多梳建立复合体 (PRC2) 的区域。被 H3K27triMe 沉默的细胞基因通过顺式 DNA 元件或通过与 YY1 或非编码 RNA (ncRNA) 相互作用间接招募 PRC2。 PRC2 然后在赖氨酸 27 处甲基化组蛋白 H3，从而导致异染色质形成。因此，我们将确定 PRC2 是否直接与 HSV-1 基因组上的元件结合，或者这种结合是否是通过其他因素介导的。异染色质建立后，第二个 PRC，即 PRC 维持复合物 (PRC1)，识别 H3K27triMe 标记并维持抑制状态。 RNA 免疫沉淀 (RIP) 数据表明，LAT 2.0kb 稳定内含子与 PRC1 结合，表明内含子隔离 PRC1 并降低 H3K27triMe 对潜在基因组的抑制程度。我们假设这种与 PRC1 结合的竞争是维持裂解基因处于更灵活的“抑制但可逆”异染色质状态以促进重新激活的关键因素。在目标 2 中，我们将表征 PRC1 与 LAT 内含子的结合，并确定减少细胞中 PRC1 的量是否足以增强 HSV 重新激活。最后，为了发生重新激活，必须释放 PRC1 抑制，并且与 HSV-1 裂解启动子相关的染色质必须重塑为转录允许状态。我们之前已经表明，在外植体诱导的小鼠潜伏神经节重新激活过程中，LAT 丰度会短暂增加。目标 3 的目标是确定如何去除 H3K27triMe 标记以使裂解转录继续进行，以及 LAT 是否直接参与此过程。拟议的研究将为 HSV-1 潜伏期裂解基因沉默机制提供关键见解，并详细介绍 LAT 内含子在调节 H3K27triMe 中所起的新作用。此外，这项工作可能为 PRC 调节细胞基因的新机制提供新的见解。