Repression and activation of persisting HSV genomes

描述（由申请人提供）：单纯疱疹病毒感染可导致复发性溃疡，导致失明的间质角膜炎或脑炎。目前处理HSV感染的临床策略以病毒的生产生命周期为目标。核苷类似物，如无环鸟苷及其衍生物，以病毒DNA复制为目标。然而，核苷类似物对潜伏状态几乎没有影响。考虑针对潜伏期和再激活方面的抗病毒方法必须考虑这些现象的机制基础。潜伏期的特点是相对于生产性感染的转录严重减少。潜伏期间的抑制是由于病毒基因组的染色质化。介导和维持病毒基因组染色质的抑制因子尚不清楚。这些可以作为再激活过程的目标。目前还不清楚哪些病毒和细胞机制参与逆转染色质的抑制作用，从而在再激活过程中恢复生产性基因转录。我们将讨论与这些问题相关的三个假设：病毒基因组受到组成性和兼性异染色质的抑制，这些染色质类型的效应物定位或结合到静止的基因组上以维持这些结构。2. 已知ICP0可以逆转染色质结构的抑制作用；然而，现有证据表明，面对表观遗传抑制，ICP4和VP16也可能具有激活转录的功能。我们认为这三种可能都能够进一步推进再激活过程。3. ICP0指定了多种有助于静止基因组抑制的活性。提出了三个相互关联的具体目标来解决这些假设：确定参与抑制静止和潜伏基因组的细胞成分。这将通过对静止和潜伏基因组进行系统的ChIP和ChIP-seq分析，并结合shRNA耗尽实验来解决。2. 确定三种病毒反激活因子，ICP4， ICP0和VP16是否以及如何在异染色质抑制的基因组上起作用。我们将把ICP0， ICP4和VP16提供给含有静止或潜伏病毒基因组的细胞，以评估这些蛋白的能力：1 .逆转特定的抑制染色质结构；招募RNA polII，激活转录。3. 确定参与抑制性染色质逆转和转录激活的ICP0的结合伙伴。ICP0的跨显性突变体将被tap标记，纯化的含ICP0复合物将通过质谱分析来鉴定复合物中所含的细胞蛋白。这些相互作用的重要性将由基因决定。预计一些鉴定的蛋白质将存在于目的1中鉴定的被抑制的静止基因组上。这些研究的完成将有助于更好地理解潜伏基因组的抑制和激活。

DESCRIPTION (provided by applicant): Herpes simplex virus infections can result in recurrent sores, stromal keratitis leading to blindness, or encephalitis. Current clinical strategies to address HSV infections target the productive life cycle of the virus. Nucleoside analogues, such as acyclovir and its derivatives, target viral DNA replication. However, nucleoside analogs have little effect on the latent state. Considerations of antiviral approaches targeting aspects of latency and reactivation must consider the mechanistic underpinnings for these phenomena. Latency is characterized by a severe reduction in transcription relative to productive infection. The repression seen during latency is due to the chromatinization the viral genome. The repressors that mediate and maintain chromatin on the viral genome are not known. These may serve as targets for the reactivation processes. It is also not known what viral and cellular mechanisms are involved in reversing the repressive effects of chromatin resulting in the resumption of productive gene transcription in the process of reactivation. We will address three hypotheses relevant to these issues: 1. Viral genomes are repressed by both constitutive and facultative heterochromatin and to be determined effectors of these chromatin types localize or bind to quiescent genomes to maintain these structures. 2. ICP0 is known to reverse the repressive effects of chromatin structure; however available evidence suggests that ICP4 and VP16 may also function to activate transcription in the face of epigenetic repression. We suggest that all three may be capable of further advancing the reactivation process. 3. ICP0 specifies multiple activities that contribute to the derepression of quiescent genomes. Three interconnected specific aims are proposed to address these hypotheses: 1. Determine the cellular components that are involved in the repression of quiescent and latent genomes. This will be addressed by a systematic ChIP and ChIP-seq analysis of quiescent and latent genomes, combined with shRNA depletion experiments. 2. Determine if and how the three viral transactivators, ICP4, ICP0 and VP16 function on genomes repressed by heterochromatin. We will provide ICP0, ICP4 and VP16 to cells harboring quiescent or latent viral genomes to assess the ability of these proteins to: i. reverse specific repressive chromatin structures, and ii. recrit RNA polII, and activate transcription. 3. Determine the binding partners of ICP0 involved in the reversal of repressive chromatin and the activation of transcription. Transdominant mutants of ICP0 will be TAP-tagged, and the purified ICP0-containing complexes will be analyzed by mass spectrometry to identify the cellular proteins contained in the complexes. The significance of these interactions will be genetically determined. It is anticipated that some of the identified proteins will be those present on repressed quiescent genomes identified in aim 1. The completion of these studies will provide a better understanding of the repression and activation of latent genomes.