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Defining the mechanism of cytomegalovirus kinase-mediated regulation of viral gene expression and the therapeutic implications.

机译：定义巨细胞病毒激酶介导的病毒基因表达调控的机制及其治疗意义。

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摘要

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that establishes lifelong infections. HCMV is an opportunistic pathogen that causes disease in immunocompromised patients and is a common agent of congenital infection. Current approved treatments options are efficacious but concerns remain about their high toxicity and the rapid development of antiviral resistance. Additionally, the available anti-HCMV agents do not target early events of infection. Therefore, identifying new treatment options that are both safe and effective is of great importance.;The HCMV kinase, pUL97 is a serine/threonine-specific kinase with multiple important roles during infection. pUL97 acts as a viral cyclin-dependent kinase (Cdk) to stimulate cell cycle progression and functions to promote viral DNA synthesis, nuclear egress, and virion maturation. In addition, pUL97 is delivered upon infection as part of the virion, though its role prior to viral gene expression had not been explored. The importance of pUL97 during infection makes it an ideal target for antiviral treatment. The pUL97-specific kinase inhibitor, maribavir (MBV) was developed and is currently used in clinics as a second line anti-HCMV agent. MBV has low toxicity, though mutations in the UL27 open reading frame result in MBV resistance. pUL27 was found to induce p21Cip1 expression and cell cycle arrest. We hypothesized that pUL97 functions to stimulate viral immediate early (IE) gene expression, that pUL97 inhibition by MBV could be used as a safe anti-HCMV agent in a cell culture model of congenital infection, and that MBV resistance was influenced by p21Cip1 and Cdk activity.;The main aims of this thesis were to characterize the molecular mechanism by which pUL97 promotes IE gene expression, assess the effectiveness of MBV in a congenital HCMV model, and identify how p21Cip1 and Cdk activity contribute to MBV resistance. We found that inhibition or deletion of pUL97 resulted in a transient decrease in IE gene expression. pUL97 was sufficient to transactivate IE expression and virion-derived pUL97 increases IE expression. Interestingly, inhibiting histone deacetylases (HDACs), which function to repress transcription, rescued IE expression under pUL97 inhibition. We observed that pUL97 activity increased histone acetylation and decreased HDAC1 association with the major IE promoter (MIEP). Further, pUL97 interacted with and increased phosphorylation of HDAC1.;Previous studies have shown that IE and late viral events contribute to defects in cell culture models of congenital HCMV infection. We therefore tested the effectiveness of MBV for inhibiting infection in the embryonic stem cell-derived neuroprogenitor cell (NPC) model of congenital infection. MBV was demonstrated to effectively limit IE gene expression, expression of other viral genes, viral DNA synthesis and viral yield. Because antiviral resistance poses a major obstruction to treating infections in clinical infections we explored the mechanism of UL27-associated MBV resistance. We found that pUL97 interacts with and promotes phosphorylation of pUL27, while also blocking pUL27- mediated induction of p21Cip1 expression. These studies also resulted in the finding that the full antiviral effect of MBV required p21Cip1 and that UL27-associated MBV resistance required Cdk activity.;Work in this thesis provides mechanistic insight into the role of the pUL97 in activation of IE gene expression, suggests that inhibiting pUL97 in NPCs effectively reduces viral replication, and provides a potential mechanism for antiviral resistance to viral kinase inhibition. II.
机译：人类巨细胞病毒（HCMV）是一种建立终生感染的无处不在的疱疹病毒。 HCMV是机会病原体，可在免疫功能低下的患者中引起疾病​​，并且是先天性感染的常见病因。当前批准的治疗方法是有效的，但是仍然关注其高毒性和抗病毒耐药性的快速发展。此外，可用的抗HCMV药物不针对感染的早期事件。因此，确定既安全又有效的新治疗方法非常重要。HCMV激酶pUL97是丝氨酸/苏氨酸特异性激酶，在感染过程中具有多种重要作用。 pUL97充当病毒细胞周期蛋白依赖性激酶（Cdk），以刺激细胞周期进程，并具有促进病毒DNA合成，核外出和病毒体成熟的功能。另外，尽管尚未探索pUL97在病毒基因表达之前的作用，但其在感染时作为病毒体的一部分被递送。 pUL97在感染期间的重要性使其成为抗病毒治疗的理想靶标。已开发出pUL97特异性激酶抑制剂maribavir（MBV），目前在临床上用作抗HCMV的二线药物。 MBV具有低毒性，尽管UL27开放阅读框中的突变会导致MBV耐药。发现pUL27诱导p21Cip1表达和细胞周期停滞。我们假设pUL97的功能是刺激病毒立即早期（IE）基因表达，MBV对pUL97的抑制作用可以用作先天性感染细胞培养模型中的安全抗HCMV药物，并且MBV耐药性受p21Cip1和Cdk的影响本论文的主要目的是鉴定pUL97促进IE基因表达的分子机制，评估先天性HCMV模型中MBV的有效性，并鉴定p21Cip1和Cdk活性如何促进MBV抗性。我们发现pUL97的抑制或删除导致IE基因表达的瞬时下降。 pUL97足以激活IE表达，而病毒体来源的pUL97可以提高IE表达。有趣的是，抑制组蛋白脱乙酰基酶（HDACs）的功能是抑制转录，在pUL97抑制下拯救了IE表达。我们观察到pUL97活性增加了组蛋白乙酰化作用，并降低了与主要IE启动子（MIEP）的HDAC1关联。此外，pUL97与HDAC1相互作用并增加其磷酸化。先前的研究表明IE和晚期病毒事件助长了先天性HCMV感染的细胞培养模型中的缺陷。因此，我们在先天性感染的胚胎干细胞衍生的神经祖细胞（NPC）模型中测试了MBV抑制感染的有效性。已证明MBV可有效限制IE基因表达，其他病毒基因的表达，病毒DNA合成和病毒产量。由于抗病毒耐药性是治疗临床感染中感染的主要障碍，因此我们探讨了UL27相关MBV耐药性的机制。我们发现pUL97与pUL27相互作用并促进其磷酸化，同时还阻断了pUL27介导的p21Cip1表达的诱导。这些研究还导致发现MBV的完全抗病毒作用需要p21Cip1，而UL27相关的MBV耐药则需要Cdk活性。本论文的工作提供了对pUL97在IE基因表达激活中作用的机制的见解。抑制NPC中的pUL97可有效减少病毒复制，并为抗病毒激酶抑制作用提供了潜在的机制。二。

著录项

  • 作者

    Bigley, Tarin Michael.;

  • 作者单位

    The Medical College of Wisconsin.;

  • 授予单位 The Medical College of Wisconsin.;
  • 学科 Biology Microbiology.;Biology Virology.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 291 p.
  • 总页数 291
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 高分子化学（高聚物）;
  • 关键词

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