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、导致失明的基质性角膜炎或脑炎。目前解决HSV感染的临床策略针对病毒的生产生命周期。核苷类似物，如阿昔洛韦及其衍生物，靶向病毒DNA复制。然而，核苷类似物对潜伏状态几乎没有影响。针对潜伏期和再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。潜伏期的特征在于相对于生产性感染而言转录的严重减少。在潜伏期看到的抑制是由于病毒基因组的染色质化。介导和维持病毒基因组上染色质的阻遏物尚不清楚。这些可以作为重新激活过程的目标。也不知道是什么病毒和细胞机制参与逆转染色质的抑制作用，导致在再活化过程中恢复生产性基因转录。我们将解决与这些问题相关的三个假设：1。病毒基因组被组成性和兼性异染色质抑制，这些染色质类型的效应子定位或结合到静止基因组以维持这些结构。2.已知ICP 0逆转染色质结构的抑制作用;然而，现有证据表明，ICP 4和VP 16也可能在面对表观遗传抑制时起激活转录的作用。我们认为，这三个可能是能够进一步推进复苏进程。3. ICP 0规定了有助于静止基因组去阻遏的多种活动。提出了三个相互关联的具体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基因组进行系统的ChIP和ChIP-seq分析，结合shRNA耗竭实验来解决。2.确定三种病毒反式激活因子ICP 4、ICP 0和VP 16是否以及如何在被异染色质抑制的基因组上发挥作用。我们将向携带静止或潜伏病毒基因组的细胞提供ICP 0、ICP 4和VP 16，以评估这些蛋白质的能力：逆转特异性抑制性染色质结构，和ii.转录RNA polII，并激活转录。3.确定参与抑制性染色质逆转和转录激活的ICP 0的结合伴侣。将对ICP 0的反式显性突变体进行TAP标记，并通过质谱法分析纯化的含ICP 0的复合物，以鉴定复合物中含有的细胞蛋白。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是存在于目的1中鉴定的受抑制的静止基因组上的那些蛋白质。这些研究的完成将使我们更好地了解潜在基因组的抑制和激活。 公共卫生相关性：存在通过充当DNA复制抑制剂靶向HSV的生产周期而不靶向潜伏期或再活化过程的抗病毒药物。HSV-1从潜伏期的再激活知之甚少。了解HSV在潜伏感染中持续存在并可以重新激活的机制，将有助于这些过程的干预。

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. PUBLIC HEALTH RELEVANCE: Antiviral drugs exist that target the productive cycle of HSV by acting as DNA replication inhibitors and do not target latency or the reactivation process. Reactivation of HSV-1 from latency is poorly understood. Understanding the mechanisms by which HSV persists in latent infection, and can reactivate, will facilitate interventions in these processes.