Role of Daxx degradation by pp71 during the human cytomegalovirus life cycle

描述（由申请人提供）：人类巨细胞病毒（HCMV）是一种重要的人类病原体，感染世界上大多数人口，在免疫功能受到抑制的患者中引起出生缺陷和严重疾病，并与免疫衰老和增殖性疾病如癌症、再狭窄和移植相关血管病变有关。原发性感染和潜伏感染的再激活都会引起HCMV疾病。HCMV pp71蛋白是抗病毒治疗的一个有吸引力的靶标，因为它是一种转录因子和细胞周期调节剂，在溶解性感染和潜伏性感染的最初阶段发挥关键作用。我们的长期目标是确定pp71在HCMV感染细胞中每种功能背后的分子机制，pp71在裂解和潜伏病毒复制周期中的不同活性的贡献，并利用这些知识设计针对pp71蛋白的HCMV抗病毒策略。pp71是一种被膜蛋白，通过感染性颗粒传递到细胞中，控制病毒即时早期（IE）基因的表达。IE基因表达启动裂解复制，并且必须沉默，以便病毒建立潜伏期。我们发表的研究表明，一种名为Daxx的细胞蛋白沉默了HCMV IE基因的表达，并且在裂解感染期间，被膜递送的pp71定位于细胞核，并以蛋白酶体依赖、泛素独立的方式降解Daxx，以刺激IE基因的表达（63,132）。我们新的初步数据表明pp71是诱导Daxx修饰型的必要和充分条件。由于这种修饰的时间与Daxx降解有关，我们将测试Daxx的这种修饰是否是Daxx降解的泛素无关信号。我们最近也发表了被膜递送的pp71被困在细胞质中，因此在体外HCMV建立静止或潜伏样感染的细胞中不能降解Daxx（133）。在这些细胞中，IE基因以daxx依赖的方式沉默。因此，HCMV感染后Daxx的稳定性决定了被感染细胞是开始裂解还是建立潜伏样感染。我们新的初步证据表明，Daxx和pp71也控制CD34+未分化骨髓细胞中建立的真正潜伏感染。由于CD34+细胞很难作为未分化细胞在培养中维持，我们正在开发胚胎干细胞作为一种更通用的模型来研究HCMV潜伏期。在这里，我们建议进一步研究pp71介导的Daxx降解在HCMV潜伏期中的作用，并确定pp71降解Daxx的分子机制。本提案基于我们已发表和未发表的发现，利用最先进的分子和遗传方法，并引入创造性和创新性的新方法来研究HCMV潜伏期。公共卫生相关性：几乎每个人都感染了人类巨细胞病毒（HCMV），一旦感染，就永远无法摆脱这种病毒。HCMV可引起年轻人、老年人、病人和其他健康患者的疾病。我们正在研究病毒如何开始复制，希望找到阻止病毒复制的方法，从而治疗HCMV疾病。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is a significant human pathogen that infects the majority of the world's population, causes birth defects and severe disease in patients with suppressed immune function, and is associated with immunosenescence and proliferative diseases such as cancer, restenosis, and transplant-associated vasculopathy. Both primary infection and reactivations of latent infections cause HCMV disease. The HCMV pp71 protein is an attractive target for antiviral therapy because it is a transcription factor and cell cycle regulator that performs critical functions at the very beginning of both lytic and latent infections. Our long- term goal is to determine the molecular mechanisms behind each function of pp71 in HCMV-infected cells, the contribution that the different activities of pp71 make during lytic and latent viral replication cycles, and to use this knowledge to design antiviral strategies against HCMV that target the pp71 protein. pp71 is a tegument protein that is delivered to cells by infectious particles and controls the expression of the viral immediate early (IE) genes. IE gene expression initiates lytic replication, and must be silenced in order for the virus to establish latency. Our published work shows that a cellular protein called Daxx silences HCMV IE gene expression, and that during a lytic infection, tegument-delivered pp71 localizes to the nucleus and degrades Daxx in a proteasome-dependent, ubiquitin-independent manner to stimulate IE gene expression (63,132). Our new preliminary data indicates that pp71 is necessary and sufficient to induce a modified form of Daxx. Because the timing of this modification correlates with Daxx degradation, we will test if this modification of Daxx is the ubiquitin-independent signal for Daxx degradation. We have also recently published that tegument-delivered pp71 is trapped in the cytoplasm and thus fails to degrade Daxx in cells where HCMV establishes a quiescent or latent-like infection in vitro (133). In these cells, IE genes are silenced in a Daxx-dependent manner. Thus, Daxx stability upon HCMV infection determines whether infected cells initiate lytic, or establish latent- like infections. Our new preliminary evidence indicates that Daxx and pp71 also control true latent infections established in CD34+ undifferentiated myeloid cells. Because CD34+ cells are difficult to maintain as undifferentiated cells in culture, we are developing embryonic stem cells as a more versatile model to study HCMV latency. Here we propose to further our examination of the role that pp71-mediated degradation of Daxx plays during HCMV latency, and to define the molecular mechanism through which pp71 degrades Daxx. This proposal builds upon our published and unpublished discoveries, utilizes state of the art molecular and genetic approaches, and introduces creative and innovative new methodologies to study HCMV latency. PUBLIC HEALTH RELEVANCE: Almost everyone is infected with human cytomegalovirus (HCMV), and once infected, can never get rid of the virus. HCMV causes disease in young, old, sick, and otherwise healthy patients. We are studying how the virus begins to replicate in the hope of finding ways to stop viral replication and thus treat HCMV disease.