Mapping the lipid envelope composition of SARS-CoV2 for reducing transmission, thrombosis and inflammation

绘制 SARS-CoV2 的脂质包膜成分图，以减少传播、血栓形成和炎症

• 批准号: BB/W003376/1
• 负责人: Valerie O'Donnell
• 金额: $ 76.96万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW003376%2F1
• 关键词: Mapping; lipid; envelope; composition; SARS

Coronaviruses are enveloped viruses, like influenza, Herpex Simplex and HIV. Thus, they are surrounded by a lipid bilayer derived from the host cell, in this case oral/lung epithelia. Surprisingly little is known about coronavirus membranes. Old studies suggest that virions bud from the phospholipid (PL)-rich ERGIC membrane, unlike many other enveloped viruses which bud from the plasma membrane, this a high level of PLs is likely. Importantly, the envelope of the SARS-CoV2 virus has not been mapped. We successfully purified SARS-CoV2 virus from Vero cell cultures on sucrose gradients, validated purity by nanosight, and are analysing its lipid composition using established mass spectrometry protocols (Stanton/O'Donnell laboratories). We can detect common PL and the full repertoire is currently being mapped and quantified. However, this now needs to be extended using human patient virus isolates since the composition is predicted to depend on the host cell and its metabolism. Notably, how virus membranes vary dependent on the host is unknown. Virus will be isolated from saliva, sputum and BAL of hospitalised patients with COVID 19. We have already obtained BAL, and subglottic aspirates, from patients intubated on ITU, and isolated live virus with titres >104 PFU/ml. Saliva will be obtained from patients using infrastructure set up as part of our ongoing clinical trial (ISRCTN25647404). We have isolated virus from saliva (104 - 105 PFU/ml) from patients on our ward. Virally-infected epithelial cells can also be harvested from the nasal mucosa using direct brushing. We plan to obtain virus from asymptomatic students donating to our university testing centre (if samples are available at that time). Virus will be purified using sucrose gradient centrifugation (already validated using Vero-derived virus). Lipids will be extracted using a chloroform/methanol method before lipidomics as described below. As this is a pilot study, we will aim for 20 BAL and 50-100 saliva samples as first step. Ethical approval processes for observational studies are well established and we anticipate no problems.A second approach will be to analyse virus cultured in A549 (human alveolar epithelial cell line transfected to overexpress ACE2 and TMPRSS2). This can be manipulated through exposure to cytokines consistent with the inflammatory status observed in COVID19 (PMID:32302401,32353870, 32678432). This model will allow scale up of virus isolation, enabling in-depth mechanistic studies to be undertaken. Virus will be cultured in A549 (+ACE2/TMPRSS2). The impact of inflammation will be tested using cytokines known to be elevated in COVID19, to mimic inflamed airway cytokine storm. These will include: IL-1b, IL-2, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-17, IFNg, IFNg-inducible protein 10, monocyte chemoattractant protein 1 (MCP1), G-CSF, macrophage inflammatory protein 1a, and TNFa (PMID:32302401,32353870, 32678432). Two other cytokines of significant interest are IL-4/IL-13, known to play a key role in allergic lung diseases like asthma. IL-4/IL-13 will be tested +/- dexamethasone. Importantly, IL-4/IL-13 are inducers of procoagulant eoxPL generation in airway epithelia (PMCID:PMC5765418). It is therefore critical that we determine the impact these cytokines have on envelope composition, to determine how this may impact dissemination of bioactive lipids via the virion envelope. This will be conducted on in vitro and in vivo isolated virus and to complement this we will also analyse the subcellular membranes of A549 cells.Third, we will conduct a transcriptomic analysis of A549 cells during infection focusing on lipid metabolism. This will determine which lipid signalling pathways are utilised/hijacked by the virus to support replication (particularly PL synthesis), aiming to identify druggable targets for intervention based on interfering with the lipid envelope. RNAseq will be performed for a time course of 3 independent experiments.

冠状病毒是有包膜的病毒，如流感、单纯疱疹病毒和艾滋病毒。因此，它们被来自宿主细胞的脂质双层包围，在这种情况下是口腔/肺上皮。令人惊讶的是，人们对冠状病毒膜知之甚少。以往的研究表明，病毒粒子从富含磷脂(PL)的ERGIC膜上发芽，不像许多其他被包裹的病毒从质膜上发芽，这可能是高水平的PL。重要的是，SARS-CoV2病毒的包膜还没有被绘制出来。我们在蔗糖梯度上成功地从Vero细胞培养物中纯化了SARS-CoV2病毒，通过Nanosight验证了纯度，并正在使用已建立的质谱学方案(Stanton/O‘Donnell实验室)分析其脂质成分。我们可以检测到常见的PL，目前正在绘制和量化完整的曲目。然而，现在需要使用人类患者病毒分离株来扩展这一点，因为预计病毒的组成取决于宿主细胞及其新陈代谢。值得注意的是，病毒膜如何根据宿主的不同而变化尚不清楚。病毒将从COVID 19住院患者的唾液、痰和BAL中分离出来。我们已经从接受ITU插管的患者身上获得了BAL和声门下抽吸物，并分离出滴度为104pfu/ml的活病毒。唾液将从患者身上获得，这些基础设施是我们正在进行的临床试验的一部分(ISRCTN25647404)。我们从我们病房患者的唾液中分离到病毒(104-105pfu/ml)。病毒感染的上皮细胞也可以通过直接刷牙从鼻黏膜中获取。我们计划从无症状的学生那里获得病毒，捐赠给我们的大学检测中心(如果当时有样本的话)。病毒将使用蔗糖梯度离心法进行纯化(已经使用Vero衍生病毒进行了验证)。脂类组学之前，将使用氯仿/甲醇方法提取脂类，如下所述。由于这是一项初步研究，我们的第一步目标是采集20份BAL和50-100份唾液样本。观察性研究的伦理审批程序已经建立得很好，我们预计不会有问题。第二种方法将分析在A549(高表达ACE2和TMPRSS2的人肺泡上皮细胞系)中培养的病毒。这可以通过接触与COVID19中观察到的炎症状态一致的细胞因子来操纵(PMID：32302401,32353870,32678432)。这一模式将使病毒分离的规模扩大，从而能够进行深入的机制研究。病毒在A549(+ACE2/TMPRSS2)上培养。炎症的影响将使用已知的COVID19中升高的细胞因子来测试，以模拟炎症的呼吸道细胞因子风暴。包括：IL-1b、IL-2、IL-6、IL-7、IL-8/CXCL8、IL-10、IL-17、干扰素、干扰素诱导蛋白10、单核细胞趋化蛋白1、粒细胞集落刺激因子、巨噬细胞炎性蛋白1a和肿瘤坏死因子α(PMID：32302401,32353870,32678432)。另外两种令人感兴趣的细胞因子是IL-4/IL-13，已知在哮喘等过敏性肺部疾病中发挥关键作用。IL-4/IL-13将检测为+/-地塞米松。重要的是，IL-4/IL-13是呼吸道上皮细胞产生促凝剂eoxPL的诱导剂(PMCID：PMC5765418)。因此，我们必须确定这些细胞因子对包膜成分的影响，以确定这可能如何影响生物活性脂类通过病毒粒子包膜的传播。这将在体外和体内分离的病毒上进行，为了补充这一点，我们还将分析A549细胞的亚细胞膜。第三，我们将对A549细胞在感染过程中进行转录分析，重点是脂代谢。这将确定哪些脂质信号通路被病毒利用/劫持以支持复制(特别是PL合成)，目的是根据干扰脂质包膜来确定干预的可用药靶点。RNAseq将进行3个独立实验的时间进程。