Cytomegalovirus manipulation of functional cortical tissue development

项目摘要 人巨细胞病毒（hCMV）感染了世界上大部分人口，并建立了一个终身的 持续性感染定义为裂解和潜伏状态，hCMV仍然是一个重要的问题， 人体健康hCMV是美国非遗传性出生缺陷的主要原因， 世界如果病毒穿过胎盘屏障，hCMV在胎儿神经祖细胞中复制， 中枢神经系统损伤存在几种抗病毒治疗方法，但未被批准用于 由于毒性和潜在的致畸作用而导致妊娠。本文所述的合作项目 开始应用，以解决在理解潜在发病机制方面的重大缺陷 先天性hCMV感染和缺乏抗病毒方法。 hCMV表达一种病毒激酶pUL 97，它调节多种病毒和细胞过程， 被抗病毒化合物Maribavir抑制。pUL 97的一个靶标对hCMV复制很重要， Maribavir活性是细胞Tip 60乙酰转移酶。Tip 60是一种肿瘤抑制因子， 许多病毒，并在组织发育过程中调节干细胞更新和分化。Tip 60是 以几种同种型表达，并且是较大的多蛋白Tip 60复合物的一部分，其中特异性功能 活性由相关蛋白决定。 本提案的目的有两个：（1）明确hCMV的影响和作用， pUL 97激酶在神经细胞分化为功能性3-羟色胺过程中调节细胞Tip 60乙酰转移酶 三维组织，和（2）评估神经组织中的hCMV激酶抑制剂Maribavir。我们假设 hCMV pUL 97操纵Tip 60以刺激病毒DNA合成，导致神经细胞凋亡的改变。 组织发育和分化细胞对Maribavir抗hCMV活性的敏感性。这将 目的1检验pUL 97对hCMV感染的贡献是 依赖于神经发生过程中Tip 60亚型的组织特异性表达;目的2 假设pUL 97激酶抑制阻止hCMV在三维人神经组织中扩散， 允许未感染的人神经祖细胞分化成星形胶质细胞的空间网络， 突触连接的皮层神经元。一个全面的细胞和分子生物学 技术结合创新的方法在组织工程和质谱为基础 成像将用于研究发育中的皮质组织中的hCMV感染。 先天性hCMV感染可能对儿童产生破坏性影响，对儿童的健康构成重大挑战。 这对父母来说是一个沉重的负担，对社会来说是一个沉重的负担。不管我们的假设是否正确， 这些研究将揭示hCMV pUL 97激酶生物学的基本方面和Tip 60对 神经发生、hCMV感染和临床相关细胞和组织类型中的抗病毒易感性。

PROJECT SUMMARY Human cytomegalovirus (hCMV) infects much of the world’s population and establishes a life-long persistent infection defined by both lytic and latent states, and hCMV continues to be a significant issue in human health. hCMV is the leading cause of non-heritable birth defects in the United States and likely around the world. If the virus crosses the placental barrier, hCMV replicates in fetal neural progenitor cells resulting in central nervous system damage. Several antiviral treatments exist but are not approved for use during pregnancy due to toxicity and potential teratogenic effects. The collaborative project described in this application was initiated to address the significant deficiencies in understanding the underlying pathogenesis of congenital hCMV infection and the lack of antiviral approaches. hCMV expresses a viral kinase, pUL97 that regulates diverse viral and cellular processes and can be inhibited by the antiviral compound, maribavir. One target of pUL97 that is important for hCMV replication and maribavir activity is the cellular Tip60 acetyltransferase. Tip60 is a tumor suppressor, manipulated by numerous viruses, and regulates stem cell renewal and differentiation during tissue development. Tip60 is expressed in several isoforms and is part of larger multiprotein Tip60 complexes where specific functional activity is determined by the associated proteins. The objectives of this proposal are twofold: (1) To define the impact of hCMV and the role of the hCMV pUL97 kinase in regulating cellular Tip60 acetyltransferase during neural cell differentiation into functional 3- dimensional tissues, and (2) to assess the hCMV kinase inhibitor maribavir in neural tissue. We hypothesize that hCMV pUL97 manipulates Tip60 to stimulate viral DNA synthesis resulting in alteration of neural tissue development and sensitization of differentiating cells to maribavir anti-hCMV activity. This will be tested in two aims: Aim 1 Test the hypothesis that the contribution of pUL97 to hCMV infection is dependent on tissue-specific expression of Tip60 isoforms during neurogenesis; and Aim 2 Test the hypothesis that pUL97 kinase inhibition prevents hCMV spread in 3-dimensional human neural tissues and allows uninfected human neural progenitor cells to differentiate into a spatial network of astrocytes and synaptically-connected cortical neurons. A comprehensive repertoire of cellular and molecular biological techniques combined with innovative approaches in tissue engineering and mass spectrometry-based imaging will be used to study hCMV infection in developing cortical tissue. Congenital hCMV infection can have devastating effects on children, pose significant challenges for parents, and continues to be a significant burden on society. Regardless of the validity of our hypothesis, these studies will uncover fundamental aspects of hCMV pUL97 kinase biology and Tip60’s contributions to neurogenesis, hCMV infection, and antiviral susceptibility in a clinically relevant cell and tissue type.