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

脂肪酸附着于流感病毒的血凝素（HA）是病毒复制所必需的蛋白质修饰。在我们之前的合作中，我们使用质谱法证明了硬脂酸酯与跨膜区域末端的半胱氨酸的位点特异性附着，以及两种棕榈酸酯与HA细胞质尾部的保守半胱氨酸的位点特异性附着。我们（和其他人）表明棕榈酸盐附着对病毒传染性的贡献最大。通过这一联合提议，我们希望确定酶，即dhhc -蛋白，催化人流感病毒HA的棕榈酰化。由于23种DHHC蛋白具有不同的，仅部分重叠的底物特异性，因此只有少数可能在肺气道细胞中酰基化HA。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，我们在转染的HeLa细胞中使用sirna筛选，并在HPA-1细胞中敲除已确定的候选dhhc，我们发现dhhc 2、5、8、15和20参与HA的酰化。如果用CRISPR/Cas9在人气道A549细胞中单独敲除这些dhhc， HA的棕榈酰化就会减少，病毒滴度被抑制约1 log，这表明几个dhhc协同作用。我们现在的目标是创建A549细胞，其中几种dhhc的表达同时被抑制，直到HA的酰化和病毒复制被更严重地抑制。我们将分析所鉴定的dhhc蛋白是否酰化各种甲型流感亚型的HA、乙型流感亚型的HA和丙型流感亚型的HEF，以及其他包膜病毒的膜蛋白。酰化的减少和病毒颗粒中透明质酸的脂肪酸模式也将通过质谱法密切监测。这将允许确定某种DHHC蛋白是否对棕榈酸酯或硬脂酸酯具有特异性。实验数据将由分子模型加以补充。已公布的DHHC 15和DHHC 20的空间结构显示，一个共价结合的脂肪酸被埋在由四个跨膜区域形成的疏水腔中。腔体的窄端由两个DHHC蛋白之间的可变氨基酸封闭；这个位置的两种氨基酸的性质决定了隧道的深度，从而决定了脂质的特异性。我们将模拟其他有趣的DHHC蛋白的疏水腔；特别是寻找具有短疏水空腔的DHHC，该空腔可容纳棕榈酸酯而不能容纳硬脂酸酯。此外，最近公布的HA跨膜区结构可能允许在已鉴定的DHHC蛋白中识别底物结合位点，以及HA中的哪些特征被DHHC蛋白识别。然后，预测将通过dhhc识别的HA中的氨基酸诱变及其随后使用反向遗传学和酰化分析的分析来验证。

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.