Metabolic regulatory mechanisms essential for Human Cytomegalovirus replication

描述(申请人提供)：几十年来，人们已经知道病毒会引起宿主细胞代谢的戏剧性变化，这些变化对病毒复制很重要。然而，其中涉及的机制在很大程度上仍然不清楚。剖析病毒代谢操纵机制的一个主要挑战是与测量活细胞中不同代谢活动相关的技术困难。我们开发了一种基于LC-MS/MS的方法来测量活细胞中的全球代谢活动。我们认为，阐明这些病毒机制将为开发新的抗病毒疗法提供肥沃的土壤。我们的结果表明，高滴度的HCMV复制需要三种代谢调节活性：调节糖酵解通量的AMP激活的激酶(AMPK)和磷酸果糖激酶-1(PFK1)，以及调节脂肪酸生物合成的乙酰辅酶A羧化酶(ACC1)。在未感染的细胞中，激活的AMPK直接抑制ACC1的活性，从而抑制脂肪酸的生物合成。我们发现，尽管激活了AMPK，但HCMV阻止了这种调控，保持了ACC1活性的增加。这会导致糖酵解和脂肪酸生物合成的戏剧性激活。为了探索这些机制，我们将：(I)阐明HCMV诱导AMPK激活的机制及其在HCMV复制中的作用；(Ii)阐明HCMV诱导的ACC1激活的机制；(Iii)阐明HCMV诱导的PFK1激活的机制及其对病毒复制的影响。通过阐明导致这些代谢活动激活的病毒机制，我们将寻找新的抗病毒靶点来对抗HCMV相关疾病，并进一步探索病毒对这些基本宿主细胞途径的操纵。 公共卫生相关性：人类巨细胞病毒(HCMV)是一种广泛存在的机会性病原体，可在各种免疫抑制人群中引起严重疾病，包括老年人、接受免疫抑制化疗的癌症患者、移植受者和艾滋病患者。巨细胞病毒也是先天性病毒感染的主要原因，发生在所有活产儿的1-2%，可导致多器官系统异常，大多数有症状的新生儿发生中枢神经系统损害。患者长期使用现有的抗巨细胞病毒治疗药物会产生毒副作用，并导致出现耐药病毒株，这凸显了额外抗巨细胞病毒治疗药物的必要性。我们提出的研究旨在阐明HCMV利用来驱动病毒粒子成分的生物合成的机制。阐明这些机制将为治疗阻断病毒复制和减轻HCMV相关疾病提供目标。

DESCRIPTION (provided by applicant): It has been known for decades that viruses induce dramatic changes to host-cell metabolism and that these changes are important for viral replication. The mechanisms involved, however, have largely remained obscure. A major challenge in dissecting the mechanisms of viral metabolic manipulation has been the technical difficulty associated with measuring diverse metabolic activities in live cells. We have developed a liquid chromatography-tandem mass spectrometry-based (LC-MS/MS) methodology to measure global metabolic activities in live cells. We propose that elucidation of these viral mechanisms will prove fertile ground for the development of novel anti-viral therapeutics. Our results indicate that three metabolic regulatory activities are required for high-titer HCMV replication; AMP-activated kinase (AMPK) and phosphofructokinase-1 (PFK1), both of which regulate glycolytic flux, and acetyl-CoA carboxylase (ACC1), which regulates fatty acid biosynthesis. In uninfected cells, activated AMPK directly inhibits ACC1 activity and thereby, fatty acid biosynthesis. We find that HCMV blocks this regulatory control, maintaining increased ACC1 activity despite activated AMPK. This results in a dramatic activation of both glycolysis and fatty acid biosynthesis. To explore these mechanisms, we will: (I) Elucidate the mechanisms of HCMV-induced AMPK activation and its role in HCMV replication; (II) Elucidate the mechanisms of HCMV- induced ACC1 activation; and (III) Elucidate the mechanisms of HCMV-induced PFK1 activation and how they contribute to viral replication. Through elucidating the viral mechanisms leading to the activation of these metabolic activities we will identify novel anti-viral targets to combat HCMV-associated disease and further explore viral manipulation of these fundamental host-cell pathways. PUBLIC HEALTH RELEVANCE: Human Cytomegalovirus (HCMV) is a widespread opportunistic pathogen that can cause severe disease in various immunosuppressed populations including the elderly, cancer patients receiving immunosuppressive chemotherapy, transplant recipients, and AIDS patients. HCMV is also the leading cause of congenital viral infection, occurring in 1-2% of all live births, which can result in multiple organ system abnormalities with central nervous system damage occurring in the majority of symptomatic newborns. Long term use of current anti-HCMV therapeutics in patients leads to toxic side effects and has resulted in the emergence of drug-resistant viral strains, highlighting the need for additional anti-HCMV therapeutics. Our proposed research aims to elucidate the mechanisms HCMV utilizes to drive the biosynthesis of virion components. Elucidating these mechanisms will present targets to therapeutically block viral replication and attenuate HCMV-associated disease.