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

描述（由申请人提供）：人巨细胞病毒（CMV）是美国非遗传性感音神经性听力损失的最常见原因。目前治疗妊娠期间或出生时感染的治疗方法未经证实且存在风险。实验性抗病毒化合物Maribavir是CMV蛋白激酶pUL 97的特异性抑制剂，毒性较低。我的建议的目的是确定pUL 97激酶介导的CMV基因表达调控背后的分子机制，并确定抑制激酶活性对CMV感染的胚胎干细胞衍生的神经元祖细胞的影响。我已经证明，抑制CMV激酶导致病毒立即早期（IE）基因的表达减少。这是CMV pUL 97激酶的新鉴定的功能。我们的实验室以前确定了CMV pUL 97激酶和细胞组蛋白脱乙酰酶1（HDAC 1）之间的相互作用。HDAC 1作为一种阻遏物，在感染早期被募集到MIE启动子。因此，我假设CMV pUL 97的激酶活性通过改变细胞HDAC 1活性来影响病毒主要立即早期启动子处的组蛋白修饰模式。我建议在感染过程中，在主要的立即早期启动子（MIEP）的组蛋白修饰的pUL 97依赖的变化进行评估。这些研究将使用人二倍体成纤维细胞启动。此外，我将确定pUL 97介导的HDAC 1在感染细胞内和MIEP定位的变化。其他疱疹病毒激酶磷酸化HDAC 1，我提出了几种方法来确定潜在的变化HDAC 1磷酸化pUL 97。我将确定这些变化对HDAC 1脱乙酰酶活性的影响。我将使用胚胎干细胞衍生的神经祖细胞系（ES-NPCs）作为CMV诱导的先天性CMV感染引起的神经损伤的模型系统来进一步这些研究。已经证明IE基因的表达改变胎儿/新生儿NPC分化。CMV IE基因调控人二倍体成纤维细胞的细胞周期和凋亡。我将通过证实我们的成纤维细胞研究中获得的主要结果来测试pUL 97在ES-NPC背景下这些早期事件中的作用。我将确定Maribavir在预防HCMV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pUL 97激酶抑制剂是否适用于治疗CMV感染的新生儿和可能存在先天性感染风险的CMV感染母亲。

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.