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，可感染世界上大多数人口，导致出生缺陷和免疫功能受到抑制的患者罹患严重疾病，并与免疫衰老和增殖性疾病（例如癌症、再狭窄和移植相关血管病）相关。原发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因子，在裂解感染和潜伏感染的初期发挥关键功能。我们的长期目标是确定 HCMV 感染细胞中 pp71 每种功能背后的分子机制、pp71 的不同活性在裂解和潜伏病毒复制周期中所做出的贡献，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 基因表达启动裂解复制，并且必须被沉默才能使病毒建立潜伏期。我们发表的研究表明，一种名为 Daxx 的细胞蛋白会沉默 HCMV IE 基因表达，并且在裂解感染期间，被膜递送的 pp71 定位于细胞核，并以蛋白酶体依赖性、不依赖于泛素的方式降解 Daxx，从而刺激 IE 基因表达 (63,132)。我们新的初步数据表明 pp71 对于诱导 Daxx 的修饰形式是必要且充分的。由于此修饰的时间与 Daxx 降解相关，因此我们将测试 Daxx 的此修饰是否是 Daxx 降解的泛素独立信号。我们最近还发表了外皮递送的 pp71 被困在细胞质中，因此无法降解 HCMV 在体外建立静止或潜伏样感染的细胞中的 Daxx (133)。在这些细胞中，IE 基因以 Daxx 依赖性方式沉默。因此，HCMV 感染后的 Daxx 稳定性决定了受感染的细胞是否引发裂解，或建立潜伏样感染。我们的新初步证据表明 Daxx 和 pp71 还控制 CD34 未分化骨髓细胞中建立的真正潜伏感染。由于 CD34+ 细胞很难在培养中维持为未分化细胞，因此我们正在开发胚胎干细胞作为研究 HCMV 潜伏期的更通用的模型。在这里，我们建议进一步研究 pp71 介导的 Daxx 降解在 HCMV 潜伏期所起的作用，并确定 pp71 降解 Daxx 的分子机制。该提案建立在我们已发表和未发表的发现的基础上，利用最先进的分子和遗传学方法，并引入了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：几乎每个人都感染过人类巨细胞病毒 (HCMV)，一旦感染，就永远无法摆脱该病毒。 HCMV 会导致年轻、年老、患病和其他健康患者患病。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.