Elucidating the role of Enolase 2 during HCMV infection

摘要 人巨细胞病毒(HCMV)是导致出生缺陷和免疫功能低下严重疾病的主要原因 个人。了解巨细胞病毒是如何操纵细胞代谢的将为我们提供对一种新兴的 宿主-病原体的相互作用可能会导致新的治疗靶点，这是一个重要的目标 目前的巨细胞病毒疗法生物利用度低，毒性升高。人巨细胞病毒引起大量 并调节多种寄主代谢基因的表达和活性。对于许多人来说 这些变化，目前尚不清楚它们如何促成成功感染。我们发现的一个基因是被诱导的 在感染过程中是神经元特异性的烯醇化酶2。这项提议的目的是研究烯醇化酶2的作用 在HCMV感染过程中通过三个特定的目的。在第一个目标中，我将研究烯醇化酶2是如何表达的 会导致病毒感染。我的初步数据显示：(I)HCMV在感染过程中诱导烯醇化酶2，以及(Ii) 这种shRNA介导的烯醇化酶2的缺失可以减轻病毒感染。ShRNA和CRISPR-Cas9介导 本提案的所有部分都将使用Enolase 2的敲除/敲除。这一目标的第一部分将 研究这种衰减的机制以及在病毒生命周期中需要烯醇化酶2的位置。在第二个 作为这一目标的一部分，我将研究烯醇化酶2对病毒感染的特异性。有三个定义明确的 人类基因组中编码的烯醇化酶同工酶。虽然这些基因是高度保守的，但文学作品 表明它们可能有生物学上的差异。我会测试一下烯醇化酶1或3或催化死亡的能力 烯醇化酶2修复人巨细胞病毒感染。在我的第二个目标中，我将研究烯醇化酶2在巨细胞病毒介导的 代谢重新编程。烯醇化酶尚未被证明在调节糖酵解中起作用，但我的初步研究 数据显示，在感染过程中，烯醇化酶2的缺失会减少葡萄糖的消耗。使用LC/MS/MS，我将 研究在病毒感染过程中，当存在烯醇化酶2或 不。目标2的第二部分将研究不同生理条件下对烯醇化酶2的需求 温度等条件。人巨细胞病毒感染可引起包括发烧在内的流感样症状。因此， 由于烯醇化酶2的稳定性较高，因此可能需要比烯醇化酶1/3更好的选择。在我的第三个目标中，我将阐明 HCMV诱导烯醇化酶2的机制。初步数据表明UL38病毒蛋白是必需的 对于烯醇化酶2的诱导。我将研究UL38包括烯醇化酶2表达的能力，并确定 这个机制。这项提议的结果将为深入了解烯醇化酶在人巨细胞病毒感染中的作用提供依据。 并将突出烯醇化酶同功体之间的生理和生物学差异。

Abstract Human cytomegalovirus (HCMV) is a leading cause of birth defects and severe illness in immunocompromised individuals. Understanding how HCMV manipulates cellular metabolism will provide insight into an emerging host-pathogen interaction that can potentially lead to novel therapeutic targets, an important goal as many current HCMV therapies have poor bioavailability and elevated toxicity profiles. HCMV induces numerous metabolic activities and modulates the expression and activity of a variety of host metabolic genes. For many of these changes, it is unclear how they contribute to successful infection. One gene that we find to be induced during infection is the neuron-specific Enolase 2. The goal of this proposal is to investigate the role of Enolase 2 during HCMV infection through three specific aims. In the first aim, I will investigate how Enolase 2 expression contributes to viral infection. My preliminary data show that (i) HCMV induces Enolase 2 during infection, and (ii) that shRNA-mediated loss of Enolase 2 attenuates viral infection. shRNA- and CRISPR-Cas9- mediated knockdown/knockout of Enolase 2 will be utilized for all parts of this proposal. The first part of this aim will investigate the mechanism of this attenuation and where in the viral lifecycle is Enolase 2 required. In the second part of this aim, I will investigate the specificity of Enolase 2 for viral infection. There are three well defined isozymes of enolase encoded in the human genome. While these genes are highly conserved, literature has shown they may have biological differences. I will test the ability of Enolase 1 or Enolase 3 or catalytically dead Enolase 2 to restore HCMV infection. In my second aim, I will investigate the role of Enolase 2 in HCMV-mediated metabolic reprogramming. Enolase has not been shown to have a role in regulating glycolysis, yet my preliminary data show that loss of Enolase 2 ablates glucose consumption during infection. Using LC/MS/MS, I will investigate changes in metabolic reprogramming that occur during viral infection when Enolase 2 is present or not. The second part of aim 2 will investigate the requirement of Enolase 2 under different physiological conditions such as temperature. HCMV infection can cause flu-like symptoms including fever. Therefore, Enolase 2 may be required over Enolase 1/3 due to its higher stability. In my third aim, I will elucidate the mechanism behind HCMV induction of Enolase 2. Preliminary data suggest that the UL38 viral protein is required for the induction of Enolase 2. I will investigate the ability of UL38 to include Enolase 2 expression and determine the mechanism. Results from this proposal will provide insight into the role of enolase during HCMV infection and will highlight physiological and biological differences between the enolase isoforms.