Role of CMV kinase in regulating infection of ESC-derived neuroprogenitor cells

描述(申请人提供)：人类巨细胞病毒(CMV)是美国最常见的非遗传性感音神经性耳聋的原因。目前治疗孕期或出生时感染的方法未经证实且有风险。实验中的抗病毒化合物maribavir是CMV蛋白激酶pUL97的特异性抑制剂，具有低毒性。我的建议的目的是确定pUL97激酶介导的CMV基因表达调控的分子机制，并确定抑制激酶活性对CMV感染的胚胎干细胞来源的神经祖细胞的影响。我已经证明，抑制CMV激酶会导致病毒即刻早期(IE)基因表达减少。这是新近发现的CMV pUL97蛋白的一种功能。我们的实验室之前发现了CMV pUL97激酶和细胞组蛋白脱乙酰基酶1(HDAC1)之间的相互作用。HDAC1作为一种抑制因子，在感染早期被招募到MIE启动子中。因此，我推测CMV pUL97的激酶活性通过改变细胞HDAC1活性来影响病毒主要直接早期启动子上的组蛋白修饰模式。我建议评估感染期间主要直接早期启动子(MIEP)上依赖pUL97的组蛋白修饰的变化。这些研究将使用人类二倍体成纤维细胞启动。此外，我将确定pUL97介导的HDAC1在感染细胞内和MIEP定位的变化。其他疱疹病毒激酶使HDAC1磷酸化，我提出了几种方法来确定pUL97对HDAC1磷酸化的潜在变化。我将确定这些变化对HDAC1脱乙酰酶活性的影响。我将使用胚胎干细胞衍生的神经前体细胞系(ES-NPC)作为先天性CMV感染引起的CMV诱导的神经损伤的模型系统来进一步研究这些研究。已证实IE基因的表达改变了胎儿/新生儿鼻咽癌的分化。巨细胞病毒IE基因调控人二倍体成纤维细胞的细胞周期和细胞凋亡。我将通过确认我们在成纤维细胞研究中获得的主要结果，在ES-NPC的背景下测试pUL97在这些早期事件中的作用。我将确定马利巴韦在预防巨细胞病毒感染对神经元发育的致病作用方面的有效性。我的研究将确定pUL97激酶抑制剂是否适合治疗CMV感染的新生儿和可能感染CMV的母亲，这些母亲有先天性感染的风险。

DESCRIPTION (provided by applicant): Human cytomegalovirus (CMV) is the most common cause of non-hereditary sensorineural hearing loss in the U.S. Current therapies to treat infection during pregnancy or upon birth are unproven and risky. The experimental antiviral compound maribavir is a specific inhibitor of the CMV protein kinase, pUL97, and exhibits low toxicity. The objective of my proposal is to identify the molecular mechanism behind pUL97 kinase-mediated regulation of CMV gene expression and determine the impact of inhibiting kinase activity on CMV infected embryonic stem cell-derived neuronal progenitor cells. I have demonstrated that inhibition of the CMV kinase resulted in decreased expression of viral immediate early (IE) genes. This is a newly identified function of the CMV pUL97 kinase. Our lab previously identified an interaction between the CMV pUL97 kinase and cellular histone deacetylase 1 (HDAC1). HDAC1 acts as a repressor and is recruited to the MIE promoter at early times during infection. Therefore, I hypothesize that the kinase activity of CMV pUL97 influences the histone modification pattern at the viral major immediate early promoter by altering cellular HDAC1 activity. I propose to evaluate the pUL97-dependent changes of histone modifications at the major immediate early promoter (MIEP) during infection. These studies will be initiated using human diploid fibroblasts. In addition, I will identify pUL97-mediated changes in HDAC1 localization within the infected cells and at the MIEP. Other herpesvirus kinases phosphorylate HDAC1 and I propose several approaches to identify potential changes in HDAC1 phosphorylation by pUL97. I will determine the impact of these changes on HDAC1 deacetylase activity. I will further these studies using an embryonic stem cell-derived neural progenitor cell line (ES-NPCs) as a model system for CMV-induced neurological damage due to congenital CMV infection. Expression of IE genes has been demonstrated to alter fetal/neonate NPC differentiation. CMV IE genes regulate cell cycle and apoptosis in human diploid fibroblasts. I will test the role of pUL97 during these early events in the context of ES-NPC's by confirming the major results that were obtained in our fibroblast studies. I will determine the effectiveness of maribavir in preventing the pathogenic effects of HCMV infection on neuronal development. My studies will determine if a pUL97 kinase inhibitor is suitable for treating CMV infected neonates and possibly CMV infected mothers at risk of congenital infection.