Repression and activation of persisting HSV genomes

描述(申请人提供)：单纯疱疹病毒感染可导致复发性溃疡、间质性角膜炎导致失明或脑炎。目前应对单纯疱疹病毒感染的临床战略以病毒的生产生命周期为目标。核苷类似物，如阿昔洛韦及其衍生物，针对病毒DNA复制。然而，核苷类似物对潜伏态的影响很小。针对潜伏期和重新激活方面的抗病毒方法的考虑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，转录严重减少。在潜伏期看到的抑制是由于病毒基因组的染色质化。调节和维持病毒基因组染色质的阻遏物尚不清楚。这些可作为重新激活过程的目标。目前也不知道是什么病毒和细胞机制参与了逆转染色质的抑制作用，从而在重新激活的过程中恢复了生产性基因转录。我们将解决与这些问题相关的三个假设：1.病毒基因组既受结构性异染色质的抑制，也受兼性异染色质的抑制，并被确定为这些染色质类型的效应分子，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效应；然而，现有的证据表明，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，所有这三项都可能进一步推进重新激活进程。3.ICP0规定了多个有助于静止基因组去抑制的活动。提出了三个相互关联的特定目标来解决这些假设：1.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的基因组进行系统的芯片和芯片序列分析，结合shRNA耗尽实验来解决。2.确定三种病毒反式激活因子ICP4、ICP0和VP16是否以及如何在异染色质抑制的基因组上发挥作用。我们将向含有静止或潜伏病毒基因组的细胞提供ICP0、ICP4和VP16，以评估这些蛋白的能力：1.逆转特定的抑制性染色质结构；2.重写RNA polII，并激活转录。3.确定参与抑制染色质逆转和转录激活的ICP0结合伙伴。ICP0的反显性突变体将被TAP标记，并将对纯化的含ICP0的复合体进行质谱分析，以鉴定复合体中包含的细胞蛋白。这些相互作用的重要性将由基因决定。预计一些已鉴定的蛋白质将是在AIM 1中鉴定的被抑制的静止基因组上存在的那些蛋白质。这些研究的完成将为更好地理解潜伏基因组的抑制和激活提供更好的理解。

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.