Molecular Genetics of HSV Reactivation

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，病毒基因组保持为环状附加体，裂解基因沉默。周期性地，一些神经元内的基因组重新激活，导致复发性临床疾病。我们研究的一个主要焦点是确定HSV-1裂解基因在潜伏期期间如何在神经元中沉默，以及这个过程在重新激活期间如何逆转。在过去的项目期间，我们的研究表明多梳阻遏复合物（PRC）作为一个重要的HSV潜伏期和再激活的表观遗传调节因子。在该提议中待检验的总体假设是，病毒调节PRC与HSV基因组的结合在以促进再激活的方式控制潜伏期期间裂解基因的抑制程度方面起关键作用。这里提出的实验将确定参与这一过程的病毒元件。在目的1中，我们将鉴定HSV-1基因组中招募多梳建立复合体（PRC 2）的区域。被H3 K27 triMe沉默的细胞基因通过顺式DNA元件或间接通过与YY 1或非编码RNA（ncRNA）的相互作用招募PRC 2。然后PRC 2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PRC 2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子介导。在异染色质形成后，第二个PRC，PRC维持复合物（PRC 1），识别H3 K27 triMe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PRC 1结合，表明内含子隔离PRC 1并降低H3 K27 triMe对潜伏基因组的抑制程度。我们假设，与PRC 1结合的这种竞争是将裂解基因维持在更灵活的“抑制但可逆”异染色质状态以促进重新激活的关键因素。在目的2中，我们将表征PRC 1与LAT内含子的结合，并确定减少细胞中PRC 1的量是否足以增强HSV再活化。最后，为了使再激活发生，PRC 1抑制必须被释放，并且与HSV-1裂解启动子相关的染色质必须重塑到转录允许状态。我们以前已经表明，在潜伏的小鼠神经节，有一个短暂的增加LAT丰度的BLT诱导的再激活。目标3的目标是确定如何去除H3 K27 triMe标记以允许裂解转录进行，以及LAT是否直接参与该过程。拟议的研究将提供关键的洞察裂解基因沉默的机制，在HSV-1潜伏期和细节的新作用，LAT内含子在调制H3 K27 triMe。此外，这项工作可能会提供新的见解，PRC的细胞基因调控的新机制。

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.