Dissection of the Functions of Herpes Simplex Virus ICPO

描述(申请人提供)：潜伏的单纯疱疹病毒(HSV)重新激活引起的疾病和痛苦仍然是一个主要的医学问题，尽管目前可用的抗病毒药物有效地减轻了疾病。发展潜伏期的特异性治疗所面临的主要问题是，在潜伏期的建立和维持的过程中，大多数病毒基因组被细胞蛋白沉默。我们不知道的是被感染的神经元沉默病毒基因组的机制。如果我们知道病毒沉默的机制，我们就可以设计出小分子药物的筛选，这些药物要么增强沉默状态并阻止重新激活，要么在抗病毒药物存在的情况下重新激活病毒复制。R37 CA78766奖助金的这次修订利用了积累的知识来解决这个问题。R37 CA78766赠款完全集中在ICP0上，ICP0是一种单纯疱疹病毒调节蛋白，在生产性感染以及在建立和维持潜伏期方面正在成为病毒基因表达的关键调节因子。简而言之，在过去的三年里，我们已经取得了一个基本的发现，ICP0在早期的生产性感染中发挥了关键作用，抑制了主要目的是沉默病毒DNA的细胞器。细胞在试图沉默HSV DNA时所使用的机制由含有HDAC-1或-2、COREST、REST和LSD1的复合体组成。尤其令人感兴趣的是，Corest和Rest被认为是非神经细胞中神经基因的抑制因子。LSD1-赖氨酸特异的脱甲基酶与HDAC1或HDAC2一起在这一过程中发挥关键作用。这种复合体被ICP0分解，被病毒蛋白激酶磷酸化，然后从细胞核中排出。HDAC1或-2/corest/rest/LSD1在高效感染细胞中抑制病毒基因表达的作用的发现提出了一个有趣的问题，即该复合体的组件是否也在潜伏期间抑制病毒基因的表达。这一假设在竞争性修订申请的目标1中得到了验证。第二个目标是确定一组针对ICP0、ICP4和ICP34.5编码基因的病毒microRNAs的作用。有人提出，但还没有得到证实，这些微型RNA在沉默关键基因方面发挥了作用，使病毒基因组在潜伏期保持沉默状态。然而，还有一种基于观察到的另一种假设，即HSV在生殖性感染过程中下调包括ICP0在内的调节蛋白的合成和功能。现有数据也支持病毒微RNA是这一调控网络的组成部分的假设。目的：1.验证HDAC-1或-2、COREST、REST和LSD1复合体通过沉默HSV基因组进入体内三叉神经节感觉神经元而建立潜伏期的假说。神经细胞含有上述复合体的成分。我们计划构建破坏这种复合体的病毒，其方式与我们已经证明在非神经细胞中破坏这种复合体的方式相同？ICP0减去病毒。在精心控制的实验中，我们计划确定破坏抑制复合体是否排除或减少潜伏期的建立。目的2：确定被报道为抑制ICP0和ICP34.5的潜在候选的microRNA是否在潜伏期的建立中发挥作用，或者它们是否抑制？(即刻早期)调节蛋白ICP0、ICP22和ICP4的合成或功能，或两者兼而有之。我们在HSV基因组的微RNA映射的区域中有大量的突变体。对于我们来说，构建不能表达微小RNA的病毒相对容易。这些突变株和恢复的野生型病毒将就它们建立和保持潜在感染的能力进行测试。这些研究将在两年的资助期内完成。我们预计，这些研究将确定控制潜伏期研究的药物开发的目标，并有助于小分子抑制剂筛选的设计。 公共卫生相关性：R37 CA78766赠款的这一修订版利用积累的知识来消除由于潜伏的单纯疱疹病毒(HSV)重新激活而造成的疾病和痛苦。发展潜伏期的特异性治疗所面临的主要问题是，在潜伏期的建立和维持的过程中，大多数病毒基因组被细胞蛋白沉默。如果我们能够证实所提出的病毒沉默的机制，我们就可以设计出小分子药物的筛选，这些药物要么增强沉默状态并阻止重新激活，要么在抗病毒药物存在的情况下重新激活病毒复制。

DESCRIPTION (provided by applicant): Diseases and suffering caused by reactivations of latent Herpes Simplex Viruses (HSV) remain a major medical problem notwithstanding effective attenuation of disease by currently available antiviral drugs. The major problem facing development of specific treatment of latency is that in the course of the establishment of latency and its maintenance most of the viral genome is silenced by cellular proteins. What we do not know is the mechanism by which the infected neuron silences the viral genome. If we knew the mechanisms by which the virus is silenced, we could devise screens for small molecule drugs that either enhance the silent state and preclude reactivation or reactivate viral replication in the presence of antiviral drugs. This revision of the R37 CA78766 grant takes advantage of accrued knowledge to address this issue. The R37 CA78766 grant focuses entirely on ICP0, a herpes simplex virus regulatory protein that is emerging as a key regulator of viral gene expression in both productive infections and in the establishment and maintenance of latency. In brief, in the past 3 years we have made a fundamental discovery that ICP0 plays a key role early in productive infection in suppressing the cellular machinery whose main objective is to silence viral DNA. The machinery employed by the cells in its attempt to silence HSV DNA consists of a complex containing HDAC-1 or -2, CoREST, REST and LSD1. Of particular interest is the fact that CoREST and REST are known as repressors of neuronal genes in non neuronal cells. LSD1 - the lysine specific demethylase along with HDAC1 or HDAC2 play a key role in this process. This complex is disassembled by ICP0, phosphorylated by viral protein kinases and expelled from the nucleus. The discovery of the role of the HDAC1 or -2/CoREST/REST/LSD1 in suppressing viral gene expression in productively infected cells raises the interesting question whether components of this complex also act as repressors of viral gene expression during latency. This hypothesis is tested in Aim 1 of the Competitive Revision Application. The objective of the second aim is to define the role of a set of viral micro RNAs that target the genes encoding ICP0, ICP4 and ICP34.5. It has been suggested, but not proven, that these micro RNAs play a role in silencing key genes to enable the maintenance of the silent state of the viral genome during latency. There is, however, an alternative hypothesis based on observations that HSV down regulates the synthesis and function of regulatory proteins including ICP0 in the course of productive infection. The available data also support the hypothesis that the viral micro RNAs are components of this regulatory network. These aims are as follows: Aim 1. To test the hypothesis that components of the HDAC-1 or -2, CoREST, REST and LSD1 complex enable establishment of latency by silencing the HSV genome upon entry into the sensory neurons of trigeminal ganglia in vivo. Neuronal cells contain components of the complex described above. We plan to construct viruses that disrupt the complex in the same way that we have shown that in non neuronal cells disruption of this complex complements ?ICP0 minus viruses. In carefully controlled experiments we plan to determine whether disruption of the suppressor complex precludes or diminishes the establishment of latency. Aim 2; To determine whether the micro RNAs reported as potential candidates for suppression of ICP0 and ICP34.5 play a role in establishment of latency or whether they suppress the synthesis or function of ? ( immediate early) regulatory proteins ICP0, ICP22 and ICP4 or both. We have a large number of mutants in the domain of the HSV genome in which the micro RNAs map. It is relatively easy for us to construct viruses that fail to express the micro RNAs. These mutants and the restored wild-type viruses will be tested with respect to their ability to establish and maintain latent infections. The studies will be completed during the two year grant period. We anticipate that these studies will identify targets for development of drugs that control latency studies and contribute to the design of screens for small molecule inhibitors. PUBLIC HEALTH RELEVANCE: This revision of the R37 CA78766 grant takes advantage of accrued knowledge to ablate the diseases and suffering caused by reactivations of latent Herpes Simplex Viruses (HSV). The major problem facing development of specific treatment of latency is that in the course of the establishment of latency and its maintenance most of the viral genome is silenced by cellular proteins. If we could substantiate the proposed mechanisms by which the virus is silenced, we could devise screens for small molecule drugs that either enhance the silent state and preclude reactivation or reactivate viral replication in the presence of antiviral drugs.