Repression and Activation of Persisting HSV Genomes

描述(申请人提供)：单纯疱疹病毒可以经历生产性感染，其中所有病毒基因都得到表达，最终导致后代病毒的产生和细胞死亡，也可以进入潜伏状态，其特征是相对缺乏病毒基因表达、基因组持久性和细胞存活。潜伏状态通常只出现在神经元中，可能涉及即刻早期(IE)基因表达的减弱，因此缺乏后来的病毒基因表达。IE蛋白ICP0已被证明有助于从潜伏状态到裂解状态的转变，从而导致典型的疱疹病毒反应。我们一直在研究不表达五种IE蛋白中任何一种的病毒在感染任何细胞时的行为。基因组在转录上是静止的，在细胞中持续存在，没有看到细胞病变效应。通过提供反式ICP0，它们可以在转录水平上被激活。因此，该系统具有与HSV潜伏期相关的一些关键特征，但也更适合于生化和分子研究。在过去的资助期里，我们发现：静止的基因组紧密地堆积在染色质中，低乙酰化组蛋白处于类似异染色质的状态，持久的基因组是环状的，在感染不表达IE基因的病毒后产生抗病毒反应。重要的是，ICP0的加入抑制和/或逆转了所有这些过程。在目前的应用中，我们建议进一步研究ICP0对HSV基因组染色质和环化的影响。提出了四个具体目标：(I)从整体结构、染色质类型和驻留在持久基因组上的特定组蛋白的特定残基的修饰状态方面进一步研究持久病毒基因组的状态，(Ii)确定ICP0如何影响特定组蛋白残基的修饰状态，以了解ICP0影响的细胞染色质重塑途径，从而可能参与重新激活，(Iii)。确定ICP0影响细胞DNA修复途径和特定蛋白，从而抑制HSV基因组的循环，以及(Iv)确定神经细胞中持久基因组的状态。

DESCRIPTION (provided by applicant): Herpes simplex virus can undergo either a productive infection, where all the viral genes are expressed culminating in the production of progeny virus and cell death, or it can enter a latent state, which is characterized by the relative lack of viral gene expression, genome persistence, and cell survival. The latent state typically occurs only in neurons, and may involve the attenuation of immediate early (IE) gene expression, and thus the lack of later viral gene expression. The IE protein ICP0 has been shown to facilitate the transition from the latent to the lytic state, thus leading to reaction episodes that are typical of .-herpesviruses. We have been investigating the behavior of viruses that do not express any of the five IE proteins upon infection of any cell. The genomes are transcriptionally quiescent, persist in cells, and cytopathic effects are not seen. They can be rendered transcriptionally active by supplying ICP0 in trans. Thus this system has some of the key features associated with HSV latency, but is also more amenable to biochemical and molecular study. Over the past funding period we have found that; quiescent genomes are tightly packed in chromatin with hypoacetylated histones in a state resembling heterochromatin, persisting genomes are circular, and an antiviral response is generated following infection with viruses that don't express IE genes. Importantly, the addition of ICP0 inhibits and/or reverses all of these processes. In the present application we propose to further study the effects of ICP0 on chromatin and circularization of the HSV genome. Four specific aims are proposed: (i.) further investigate the state of persisting viral genomes with respect to overall structure, type of chromatin, and the modification state of specific residues of specific histones residing on persisting genomes, (ii.) determine how ICP0 affects the modification state of specific histone residues to gain an understanding of the cellular chromatin remodeling pathways affected by ICP0, and hence potentially involved in reactivation, (iii.) determine the cellular DNA repair pathways and specific proteins that are affected by ICP0, thus inhibiting the circularization of the HSV genome, and (iv.) determine the state of persisting genomes in neuronal cells.