Palmitoylation of Influenza virus hemagglutinin: search for DHHC-acyltransferases in human airway cells as potential targets for new antiviral drugs.

流感病毒血凝素的棕榈酰化：寻找人气道细胞中的 DHHC-酰基转移酶作为新抗病毒药物的潜在靶标。

• 批准号: 429543273
• 负责人: Privatdozent Dr. Michael Veit
• 金额: --
• 依托单位: Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/429543273?language=en
• 关键词: Palmitoylation; Influenza; virus; hemagglutinin; search

Attachment of fatty acids to the hemagglutinin (HA) of Influenza virus is an essential protein modification required for viral replication. In our previous collaboration we used mass spectrometry to demonstrate site-specific attachment of stearate to a cysteine at the end of the transmembrane region and of two palmitates to conserved cysteines in the cytoplasmic tail of HA. We (and others) showed that palmitate attachment contributes the most to viral infectivity. With this joint proposal we want to identify the enzymes, i.e. DHHC-proteins that catalyse palmitoylation of HA of human Influenza viruses. Since 23 DHHC proteins with distinct, only partly overlapping substrate specificities are present in humans, only a few of them might acylate HA in airway cells of the lung. We hypothesize that these DHHC-proteins are promising drug targets since their blockade will result in suppression of viral replication, while acylation of cellular proteins will not be (or very little) compromised. In our preliminary work using siRNAs screens in transfected HeLa cells and knock-out of the identified candidate DHHCs in HPA-1 cells we identified DHHCs 2, 5, 8, 15 and 20 to be involved in acylation of HA. If these DHHCs are knocked-out individually with CRISPR/Cas9 in human airway A549 cells palmitoylation of HA is reduced and virus titers are supressed by ~ one log, indicating that several DHHCs work synergistically. We now aim to create A549 cells where the expression of several DHHCs is inhibited simultaneously until HA´s acylation and virus replication is more severely inhibited. We will analyse whether the identified DHHC-proteins acylate HA of various Flu A subtypes, HA of Flu B and HEF of Flu C and also membrane proteins of other enveloped viruses. Reduction in acylation and the fatty acid pattern of HA in virus particles will be also closely monitored by mass spectrometry. This will allow determining whether a certain DHHC protein is specific for palmitate or stearate.Experimental data will be supplemented by molecular modelling. The published spatial structure of DHHC 15 and 20 revealed a covalently bound fatty acid buried in a hydrophobic cavity formed by four transmembrane regions. The narrow end of the cavity is closed by two between DHHC proteins variable amino acids; the nature of the two amino acids at this position determines the deepness of the tunnel and thus the lipid specificity. We will model the hydrophobic cavity of other interesting DHHC proteins; especially search for a DHHC with a short hydrophobic cavity that accommodates palmitate but not stearate. Furthermore, the recently published structure of the transmembrane region of HA might allow identifying the substrate binding site in the identified DHHC proteins and which features in HA are recognized by a DHHC protein. Predictions will then be verified by mutagenesis of amino acids in HA recognized by DHHCs and their subsequent analysis using reverse genetics and acylation analysis.

脂肪酸与流感病毒血凝素（HA）的连接是病毒复制所需的重要蛋白质修饰。在我们以前的合作中，我们使用质谱法来证明硬脂酸酯与跨膜区末端的半胱氨酸和两种棕榈酸酯与HA胞质尾中的保守半胱氨酸的位点特异性连接。 我们（和其他人）表明，棕榈酸酯附着对病毒感染性的贡献最大。通过该联合提案，我们希望鉴定催化人流感病毒HA棕榈酰化的酶，即DHHC蛋白。由于人体中存在23种具有不同、仅部分重叠的底物特异性的DHHC蛋白，因此其中只有少数可能使肺气道细胞中的HA酰化。我们假设这些DHHC-蛋白是有希望的药物靶点，因为它们的阻断将导致病毒复制的抑制，而细胞蛋白的酰化不会（或很少）受到损害。在我们的初步工作中，使用转染的HeLa细胞中的siRNA筛选和HPA-1细胞中鉴定的候选DHHC的敲除，我们鉴定了DHHC 2、5、8、15和20参与HA的酰化。如果在人气道A549细胞中用CRISPR/Cas9单独敲除这些DHHC，则HA的棕榈酰化降低，并且病毒滴度被抑制约一个对数，这表明几种DHHC协同工作。我们现在的目标是创建A549细胞，其中几种DHHC的表达同时受到抑制，直到HA的酰化和病毒复制受到更严重的抑制。我们将分析鉴定的DHHC蛋白是否酰化各种甲型流感亚型的HA、乙型流感B的HA和丙型流感的HEF以及其他包膜病毒的膜蛋白。还将通过质谱法密切监测病毒颗粒中HA的酰化减少和脂肪酸模式。这将允许确定某种DHHC蛋白质是否对棕榈酸酯或硬脂酸酯具有特异性。已发表的DHHC 15和20的空间结构揭示了埋在由四个跨膜区形成的疏水腔中的共价结合的脂肪酸。空腔的窄端由DHHC蛋白质之间的两个可变氨基酸封闭;该位置处的两个氨基酸的性质决定了隧道的深度，从而决定了脂质特异性。我们将模拟其他有趣的DHHC蛋白的疏水腔，特别是寻找一个DHHC与一个短的疏水腔，可容纳棕榈酸，但不硬脂酸。此外，最近公布的HA跨膜区的结构可能允许鉴定所鉴定的DHHC蛋白中的底物结合位点以及HA中的哪些特征被DHHC蛋白识别。然后，通过DHHC识别的HA中氨基酸的诱变及其随后的使用反向遗传学和酰化分析的分析来验证预测。