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）：单纯疱疹病毒类型1（HSV-1）在感觉神经元内建立了终身的潜在感染。在潜伏期期间，病毒基因组保持为圆周发散，裂解基因被沉默。定期将一些神经元内的基因组重新激活，导致复发性临床疾病。我们研究的主要重点是确定在潜伏期期间神经元中的HSV-1裂解基因如何沉默，以及在重新激活过程中如何逆转此过程。在过去的项目期间，我们的研究将Polycomb抑制剂复合物（PRC）视为HSV潜伏期和重新激活的重要表观遗传调节剂。该提案中要检验的总体假设是，PRC与HSV基因组的病毒调节在控制潜伏期抑制裂解基因的程度方面起着关键作用，以促进重新激活的方式。这里提出的实验将定义此过程中涉及的病毒元素。在AIM 1中，我们将确定募集PolyComb建立综合体（PRC2）的HSV-1基因组区域。细胞基因通过CIS DNA元素或通过与YY1或非编码RNA（NCRNA）的相互作用而被H3K27Trime募集PRC2沉默。然后，PRC2然后在赖氨酸27处甲基甲基化组蛋白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基因组上的元素结合，或者该结合是否是通过其他因素介导的。建立异染色质后，PRC维护复合物（PRC1）识别H3K27Trime标记并保持压抑状态。 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并降低了对潜在基因组的H3K27Trime抑制程度。我们假设与PRC1结合的这种竞争是维持更灵活的“抑制但可逆”异态状态促进重新激活的裂解基因的关键因素。在AIM 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的量是否是为了增强HSV重新激活的辅助。最后，为了进行重新激活，必须释放PRC1抑制作用，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。我们先前已经表明，在露天引起的潜在鼠神经节重新激活期间，纬度丰度瞬时增加。 AIM 3的目标是确定如何删除H3K27Trime标记，以允许裂解转录进行，以及LAT是否直接参与此过程。拟议的研究将为HSV-1潜伏期期间裂解基因沉默的机制提供重要的见解，以及有关LAT内含子在调节H3K27Trime中发挥的新作用的细节。此外，这项工作可能会提供有关PRC调节细胞基因的新机制的新见解。