Interplay between cellular bioenergetics and vaccinia virus infection

 描述(申请人提供)：痘苗病毒，典型的痘病毒，在根除天花的里程碑式的疫苗接种运动中使用。在疫苗接种后的时代，猴痘病毒正在成为一种严重的人类病原体，人们仍然担心天花可能被生物恐怖分子利用。然而，随着痘病毒作为重组疫苗和溶瘤治疗的有效工具的发展，这些病毒再次被视为有希望的光明。为了开发合理、有针对性的治疗方法来治疗暴露于痘病毒期间可能出现的并发症，需要对痘病毒生命周期的错综复杂的情况有更深入的了解。痘病毒只在受感染细胞的细胞质内复制。这种不同寻常的物理独立性伴随着遗传的复杂性：大约200种病毒蛋白调控病毒的进入、基因表达、基因组复制和成熟以及病毒粒子的组装和输出。尽管有这种遗传自主性，但细胞生物学过程和病毒生命周期进展之间的密切关系越来越明显。正是痘苗病毒感染和细胞的生物能量状态之间的相互作用，才是R21应用的焦点。我们的初步数据表明，病毒感染依赖于棕榈酸酯的合成和线粒体输入，以支持TCA循环和ATP的产生。此外，感染迅速导致细胞耗氧率(OCR)增加~2倍，OCR是衡量ATP产生的直接指标。尽管当脂肪酸合成减少时，生命周期的许多阶段显示出中等程度的损害，但病毒粒子组装似乎受到最严重的影响。病毒小体的结构和功能明显受损，病毒小体是高浓度的可溶性蛋白质的储存库，预定包含在病毒粒子内部。病毒质似乎分散成聚集的片段，无法与新生的病毒膜建立适当的联系。该提案分为两个相辅相成的目标：目标一：痘苗病毒感染如何调节脂肪酸合成和线粒体功能？A：在牛痘感染期间，脂肪酸的合成和利用是如何调节的？B：感染期间线粒体活性和ATP生成是如何增强的？目的II：为什么病毒粒子组装依赖于ATP的产生？这一目标将检验关于ATP作用的两个假设：是否需要ATP来确保病毒蛋白的磷酸化，这是病毒质稳定和与新月膜相关联所必需的？是否需要ATP来支持细胞伴侣(如HSP90)与病毒质蛋白的结合，以确保它们正确的折叠？所获得的洞察力将阐明痘病毒/宿主相互作用的新方面，并为抗痘病毒治疗确定新的细胞靶点。此外，深入了解牛痘如何操纵细胞生物能量环境，以及伴侣或蛋白激酶如何调节高浓度蛋白质复合体的溶解性，应该对癌症和神经退行性疾病的研究具有广泛的相关性。

 DESCRIPTION (provided by applicant): Vaccinia virus, the prototypic poxvirus, was used in the landmark vaccination campaign that eradicated smallpox. In the post-vaccination era, monkeypox virus is emerging as a serious human pathogen, and concern about the possible bioterrorist use of variola remains. With the development of poxviruses as recombinant vaccines and effective tools for oncolytic therapy, however, these viruses are once again seen in a promising light. In order to develop rational, targeted therapeutics to treat complications that may arise during exposure to poxviruses, a deeper understanding of the intricacies of the poxvirus life cycle is needed. Poxviruses replicate solely within the cytoplasm of the infected cell. This unusual physical autonomy from the nucleus is accompanied by genetic complexity: ~200 viral proteins regulate viral entry, gene expression, genome replication and maturation, and virion assembly and egress. Despite this genetic autonomy, the close relationship between cell biological processes and the progression of the viral life cycle is increasingly clear. It is he interplay between vaccinia virus infection and the bioenergetic status of the cell that is the focu of this R21 application. Our preliminary data indicate that viral infection depends upon the synthesis and mitochondrial import of palmitate to fuel the TCA cycle and ATP production. Moreover, infection leads rapidly to a ~2-fold increase in the cellular oxygen consumption rate (OCR), a direct measure of ATP production. Although many phases of the life cycle show moderate impairment when fatty acid synthesis is reduced, it is virion assembly that appears to be the most severely impacted. There is a clear impairment of the structure and function of the virosomes, which are the depots of high concentrations of soluble proteins destined for inclusion in the virion interior. The viroplasm appears to become dispersed into aggregated fragments which fail to make appropriate associations with the nascent viral membranes. The proposal is organized into two complementary aims: Aim I: How does vaccinia virus infection modulate fatty acid synthesis and mitochondrial function? A: How are the synthesis and utilization of fatty acids regulated during vaccinia infection? B: How are mitochondrial activity and ATP generation enhanced during infection? Aim II: Why is virion assembly reliant on ATP generation? This aim will test two hypotheses regarding the role of ATP: Is ATP required to ensure the phosphorylation of viral proteins needed for viroplasm stability and association with crescent membranes? Is ATP needed to support the association of cellular chaperones (such as HSP90) with viroplasmic proteins to ensure their proper folding? The insights gained should illuminate a new aspect of poxvirus/host interaction and identify new cellular targets for anti-poxviral therapy Moreover, a deeper understanding of how vaccinia manipulates the cellular bioenergetic environment, and how chaperones or protein kinases regulate the solubility of highly concentrated protein complexes, should be of broad relevance to the study of cancer and neurodegenerative diseases.