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

冠状病毒是包膜病毒，如流感病毒、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分子层所包围，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。以前的研究表明，病毒粒子是从富含磷脂（PL）的ERGIC膜中萌发的，不像许多其他包膜病毒是从质膜中萌发的，这可能是高水平的PLs。重要的是，SARS-CoV2病毒的包膜尚未被绘制出来。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，通过纳米视觉验证了纯度，并使用已建立的质谱协议（Stanton/O'Donnell实验室）分析了其脂质组成。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物进行扩展，因为预计其组成取决于宿主细胞及其代谢。值得注意的是，病毒膜如何随宿主而变化尚不清楚。将从COVID - 19住院患者的唾液、痰液和BAL中分离病毒。我们已经从插管ITU的患者中获得BAL和声门下吸出物，并分离出滴度为bb0 - 104 PFU/ml的活病毒。唾液将使用作为我们正在进行的临床试验（ISRCTN25647404）的一部分设置的基础设施从患者中获取。我们从病房病人的唾液中分离出病毒（104 - 105 PFU/ml）。病毒感染的上皮细胞也可以通过直接刷牙从鼻黏膜上收集。我们计划从捐赠给我校检测中心的无症状学生身上获取病毒（如果当时有样本）。将使用蔗糖梯度离心纯化病毒（已使用vero衍生病毒验证）。在脂质组学之前，将使用氯仿/甲醇方法提取脂质，如下所述。由于这是一项试点研究，我们的目标是20个BAL和50-100个唾液样本作为第一步。观察性研究的伦理审批程序已经建立，我们预计不会出现问题。第二种方法是分析在A549（转染过表达ACE2和TMPRSS2的人肺泡上皮细胞系）中培养的病毒。这可以通过暴露于与covid - 19中观察到的炎症状态一致的细胞因子来操纵（PMID:32302401,32353870, 32678432）。该模型将允许扩大病毒分离的规模，从而能够进行深入的机制研究。病毒将在A549 （+ACE2/TMPRSS2）中培养。将使用已知在covid - 19中升高的细胞因子来测试炎症的影响，以模拟发炎的气道细胞因子风暴。这些将包括：IL-1b、IL-2、IL-6、IL-7、IL-8/CXCL8、IL-10、IL-17、IFNg、IFNg诱导蛋白10、单核细胞趋化蛋白1 （MCP1）、G-CSF、巨噬细胞炎症蛋白1a和TNFa （PMID:32302401、32353870、32678432）。另外两种重要的细胞因子是IL-4/IL-13，已知在过敏性肺部疾病如哮喘中起关键作用。检测IL-4/IL-13 +/-地塞米松。重要的是，IL-4/IL-13是气道上皮促凝剂eoxPL生成的诱导剂（PMCID:PMC5765418）。因此，我们确定这些细胞因子对包膜组成的影响是至关重要的，以确定这可能如何影响生物活性脂质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，我们将对感染过程中的A549细胞进行转录组学分析，重点关注脂质代谢。这将确定哪些脂质信号通路被病毒利用/劫持来支持复制（特别是PL合成），旨在确定基于干扰脂质包膜的可药物干预靶点。RNAseq将连续进行3次独立实验。

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.