Molecular Genetics of HSV Reactivation

HSV 再激活的分子遗传学

• 批准号: 8187898
• 负责人: David C. Bloom
• 金额: $ 41.01万
• 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/8187898
• 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. PUBLIC HEALTH RELEVANCE: Herpes simplex virus (HSV) causes cold sores and other serious disease in humans including encephalitis and blindness. While there are antiviral drugs to treat herpes, they don't completely block the infection and there is no cure. This research is aimed at studying a cellular protein complex, PRC, involved in controlling HSV genes during latent infection and could result in identifying new targets to develop better therapies for treating herpes infections.

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身潜伏感染。在潜伏期间，病毒基因组以环状发作的形式维持，裂解基因被沉默。一些神经元内的基因组周期性地重新激活，导致复发性临床疾病。我们研究的一个主要焦点是确定HSV-1裂解基因在潜伏期如何在神经元中沉默，以及在再激活过程中如何逆转这一过程。在过去的项目期间，我们的研究表明，多梳抑制复合体（PRC）是HSV潜伏期和再激活的重要表观遗传调节剂。本提案需要验证的总体假设是，病毒调节PRC与HSV基因组的结合在控制潜伏期间裂解基因的抑制程度方面发挥关键作用，从而促进再激活。这里提出的实验将定义参与这一过程的病毒元素。在目的1中，我们将确定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 （ncRNAs）的相互作用间接募集PRC2。PRC2随后在赖氨酸27处甲基化组蛋白H3，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，或者这种结合是否通过其他因素介导。在异染色质建立第二个PRC后，PRC维持复合体（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的稳定内含子与PRC1结合，表明内含子隔离了PRC1，降低了H3K27triMe对潜伏基因组的抑制程度。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”的异色状态以促进再激活的关键因素。在Aim 2中，我们将描述PRC1与LAT内含子的结合，并确定减少细胞中PRC1的数量是否足以增强HSV的再激活。最后，为了发生再激活，PRC1抑制必须被释放，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。我们之前已经证明，在外植体诱导的潜伏小鼠神经节再激活过程中，LAT丰度会短暂增加。Aim 3的目标是确定H3K27triMe标记是如何被移除以允许裂解转录进行的，以及LAT是否直接参与这一过程。这些研究将对HSV-1潜伏期裂解基因沉默的机制和LAT内含子在调节H3K27triMe中所起的新作用提供关键的见解。此外，这一工作可能为PRC调控细胞基因的新机制提供新的见解。