Elucidating the role of Enolase 2 during HCMV infection

阐明烯醇化酶 2 在 HCMV 感染过程中的作用

• 批准号: 10556314
• 负责人: Isreal Moreno
• 金额: $ 3.14万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10556314
• 关键词: Address; Affect; Attenuated; Biochemical Pathway; Biological; Biological Availability; CRISPR/Cas technology; Cells; Congenital Abnormality; Consumption; Cytomegalovirus; Data; Development; Enzymes; Fever; Fibroblasts; Gene Expression; Genes; Glucose; Glycolysis; Goals; Heat-Shock Response; Herpesviridae; Human; Human Genome; Immune; Immunocompromised Host; Individual; Infection; Isoenzymes; Knock-out; Lead; Life Cycle Stages; Light; Literature; Mediating; Metabolic; Metabolic Pathway; Metabolism; Methods; Neuron-Specific Enolase; Neurons; Outcome; Pathogenesis; Pathway interactions; Physiological; Play; Protein Isoforms; Proteins; Regulation; Research; Resistance; Role; Specificity; Symptoms; Temperature; Testing; Therapeutic Intervention; Tissues; Toxic effect; Vaccines; Viral; Viral Proteins; Virion; Virus; Virus Diseases; Work; attenuation; base; cellular engineering; enolase; experimental study; flu; insight; knock-down; liquid chromatography mass spectrometry; metabolomics; mutant; new therapeutic target; novel; novel therapeutics; pathogen; penis foreskin; small hairpin RNA; therapeutic target

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.

抽象的 人类巨细胞病毒 (HCMV) 是免疫功能低下者出生缺陷和严重疾病的主要原因 个人。了解 HCMV 如何操纵细胞代谢将有助于深入了解新兴的 宿主与病原体的相互作用可能会导致新的治疗靶点，这是许多人的一个重要目标 目前的 HCMV 疗法生物利用度较差且毒性较高。 HCMV 诱导大量 代谢活动并调节多种宿主代谢基因的表达和活性。对于许多 这些变化，目前尚不清楚它们如何有助于成功感染。我们发现一种被诱导的基因 感染期间是神经元特异性烯醇化酶 2。本提案的目标是研究烯醇化酶 2 的作用 HCMV 感染期间通过三个具体目标。第一个目标是研究 Enolase 2 的表达方式 有助于病毒感染。我的初步数据显示 (i) HCMV 在感染期间诱导烯醇化酶 2，并且 (ii) shRNA 介导的烯醇化酶 2 缺失可减弱病毒感染。 shRNA 和 CRISPR-Cas9 介导 Enolase 2 的敲低/敲除将用于本提案的所有部分。该目标的第一部分将 研究这种减弱的机制以及病毒生命周期中的哪个阶段需要 Enolase 2。在第二个 作为这一目标的一部分，我将研究 Enolase 2 对病毒感染的特异性。有3个明确定义的 人类基因组中编码的烯醇酶同工酶。虽然这些基因高度保守，但文献报道 表明它们可能存在生物学差异。我将测试烯醇化酶1或烯醇化酶3或催化死亡的能力 烯醇化酶 2 可恢复 HCMV 感染。在我的第二个目标中，我将研究烯醇化酶 2 在 HCMV 介导中的作用 代谢重编程。烯醇酶尚未被证明具有调节糖酵解的作用，但我的初步研究 数据显示，Enolase 2 的缺失会减少感染期间的葡萄糖消耗。使用 LC/MS/MS，我将 研究当烯醇化酶 2 存在时，病毒感染期间发生的代谢重编程的变化，或 不是。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。 HCMV 感染可引起流感样症状，包括发烧。所以， 由于烯醇酶 2 具有更高的稳定性，因此可能比烯醇酶 1/3 更需要烯醇酶 2。在我的第三个目标中，我将阐明 HCMV 诱导烯醇化酶 2 背后的机制。初步数据表明需要 UL38 病毒蛋白 用于诱导烯醇酶 2。我将研究 UL38 包含烯醇酶 2 表达的能力并确定 的机制。该提案的结果将深入了解烯醇化酶在 HCMV 感染过程中的作用 并将突出烯醇酶亚型之间的生理和生物学差异。