Membrane and lipidome dynamics during enterovirus infection

摘要/摘要 肠道病毒感染是全球范围内的重大公共卫生负担。近1500万例肠道病毒感染 每年都会发生。虽然感染主要表现为症状轻微的疾病，但某些肠道病毒株可能 在儿童中，严重的呼吸道和神经系统疾病的发病率较高。目前没有 抗病毒治疗以阻止肠道病毒感染。所有正链RNA病毒，甚至无包膜病毒 脊髓灰质炎病毒（PV）样肠道病毒需要宿主膜进行增殖。这些病毒重塑宿主 脂质体以产生具有独特磷脂组成的病毒诱导膜，从而赋予独特的 这些膜上的生化和生物物理特性使其具有不同的生物功能。PV- 相关的肠道病毒和可能的许多其它病毒属于这一类。值得注意的是，在PV感染期间， 感染RNA的翻译足以在基因组复制之前诱导细胞转化，或 宿主转录启动。这一观察结果表明，转录后和/或翻译后 在哺乳动物细胞的细胞质中存在的机制，以重新编程磷脂生物合成和膜 感染后几分钟的生物合成和“枢纽”，可以增选PV，控制这些机制。 我们的研究旨在阐明调节细胞膜生物发生、功能和细胞运输的机制 通过了解PV如何选择这些机制。PV 3CD在感染期间诱导多种磷脂。 为了研究其机制，我们在HeLa细胞中表达了标记的3CD，并利用该标记分离了3CD- 相关蛋白质我们用质谱鉴定了3个CD相关蛋白。我们把数据 通过基因筛选和已知相互作用的公开数据， 诱导通过评估蛋白质与两个或三个分离度的相互作用， 根据经验，出现了c-Fos。研究已经证明c-Fos在激活酶中的直接作用， 在磷脂生物合成和膜生物发生中进行限速步骤。我们假设PV 调节c-Fos形成独特的磷脂组成的PV诱导膜， 脂质生物合成途径。因此，目标1将评价PV介导的c-Fos调节对诱导 磷脂生物合成和膜生物发生。此外，我们的初步数据评估， PV感染的细胞的脂质组显示非常长链脂肪酸合成的诱导。只有一种异构体， 极长链脂肪酸延伸酶家族（EIFVL4）产生这些脂肪酸。我们 假设PV病毒因子劫持细胞途径（即pVVL 4）产生长链脂肪酸， 在感染期间稳定含病毒囊泡。因此，目标2将描述动力学，机制， 以及PV感染过程中宿主脂质体重构的功能。这些研究不仅将阐明 在病毒感染期间控制膜动力学，而且还提供了对这些途径如何 在正常的细胞生理过程中受到调节，这为开发抗病毒药物提供了有吸引力的候选靶点。

Summary/Abstract Enterovirus infections are a significant public health burden worldwide. Nearly 15 million enterovirus infections occur each year. While infection predominantly elicits mild-symptom disease, some enterovirus strains may confer severe respiratory and neurological illnesses with higher morbidity in children. There are no current antiviral therapeutics to thwart enteroviral infections. All positive-strand RNA viruses, even non-enveloped poliovirus (PV)-like enteroviruses, require host membranes for multiplication. These viruses remodel the host lipidome to create virus-induced membranes with unique phospholipid composition, thereby conferring unique biochemical and biophysical properties upon these membranes that enable distinct biological functions. PV- related enteroviruses and likely many other viruses fall into this category. Remarkably, during PV infection, translation of the infecting RNA is sufficient to induce cellular transformations before genome replication or host transcription is engaged. This observation suggests that post-transcriptional and/or post-translational mechanisms exist in the mammalian cell cytoplasm to reprogram phospholipid biosynthesis and membrane biogenesis minutes after infection and that “hubs,” which can be co-opted by PV, control these mechanisms. Our research aims to illuminate mechanisms regulating membrane biogenesis, function, and trafficking in cells by understanding how PV co-opts these mechanisms. PV 3CD induces multiple phospholipids during infection. To investigate the mechanism, we expressed a tagged 3CD in HeLa cells and exploited the tag to isolate 3CD- associated proteins. We identified 3CD-associated proteins using mass spectrometry. We combined our data with genetic screens and published data on known interactions to arrive at a hypothetical model for lipid induction. By evaluating proteins with two or three degrees of separation from interactions discovered empirically, c-Fos appeared. Studies have demonstrated a direct role of c-Fos in activating enzymes that perform rate-limiting steps in phospholipid biosynthesis and membrane biogenesis. We hypothesize that PV modulates c-Fos to form PV-induced membranes of unique phospholipid composition by exploiting multiple lipid biosynthetic pathways. Thus Aim 1 will evaluate PV-mediated c-Fos modulation for the induction of phospholipid biosynthesis and membrane biogenesis. Furthermore, our preliminary data evaluating the lipidome of PV-infected cells shows the induction of very long-chain fatty acids synthesis. Only one isoform in the Elongation of Very Long-Chain Fatty Acids family of enzymes (ELOVL4) produces these fatty acids. We hypothesize that PV viral factors hijack cellular pathways (namely ELOVL4) to produce long fatty acids that stabilize virus-containing vesicles during infection. Thus Aim 2 will characterize the dynamics, mechanisms, and functions of host-lipidome remodeling during PV infection. These studies will not only elucidate pathways governing membrane dynamics during viral infection but also provide insight into how these pathways are regulated during normal cellular physiology—presenting attractive target candidates for developing antivirals.