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

描述（由申请人提供）：人巨细胞病毒（HCMV）是一种重要的人类病原体，感染世界上大多数人口，导致免疫功能受抑制患者的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）相关。原发感染和潜伏感染的再激活都引起HCMV病。HCMV pp71蛋白是抗病毒治疗的一个有吸引力的靶点，因为它是一种转录因子和细胞周期调节因子，在裂解和潜伏感染的最初阶段起关键作用。我们的长期目标是确定pp71在HCMV感染细胞中的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，并利用这些知识来设计靶向pp71蛋白的抗HCMV的抗病毒策略。pp71是被膜蛋白，其通过感染性颗粒递送至细胞并控制病毒立即早期（IE）基因的表达。IE基因表达启动裂解性复制，并且必须被沉默以使病毒建立潜伏期。我们已发表的研究表明，一种名为Daxx的细胞蛋白沉默HCMV IE基因的表达，在裂解性感染过程中，被膜递送的pp71定位于细胞核，并以蛋白酶体依赖性、泛素非依赖性的方式降解Daxx，刺激IE基因的表达（63，132）。我们的新的初步数据表明，pp71是必要的，足以诱导一个修改形式的Daxx。由于这种修饰的时间与Daxx降解相关，我们将测试Daxx的这种修饰是否是Daxx降解的泛素非依赖性信号。我们最近还发表了被膜递送的pp71被捕获在细胞质中，因此不能降解细胞中的Daxx，其中HCMV在体外建立静止或潜伏样感染（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的稳定性决定了感染细胞是否开始裂解或建立潜伏样感染.我们的新的初步证据表明，Daxx和pp71也控制真正的潜伏感染建立在CD34+未分化的骨髓细胞。由于CD34+细胞在培养中难以维持为未分化细胞，我们正在开发胚胎干细胞作为研究HCMV潜伏期的更通用模型。在这里，我们建议进一步研究pp71介导的Daxx降解在HCMV潜伏期中发挥的作用，并确定pp71降解Daxx的分子机制。该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，就永远无法摆脱病毒。HCMV在年轻、年老、患病和其他健康患者中引起疾病。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.