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

在流感病毒的血凝素(HA)上附着脂肪酸是病毒复制所必需的蛋白质修饰。在我们之前的合作中，我们使用质谱仪证明了硬脂酸与跨膜区末端的半胱氨酸以及两个棕榈酸酯与透明质酸细胞质尾部保守的半胱氨酸的特异性结合。我们(和其他人)表明，棕榈酸酯附着对病毒传染性的贡献最大。通过这一联合提议，我们希望确定催化人类流感病毒HA棕榈酰化的酶，即DHHC-蛋白。由于23种DHHC蛋白具有不同的、仅部分重叠的底物特异性，在人类中存在，其中只有一小部分可能使肺的呼吸道细胞中的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抑制病毒复制，而细胞蛋白的酰化不会(或很少)受到影响。在我们的初步工作中，利用siRNAs在转染的HeLa细胞中筛选，并在HPA-1细胞中敲除已鉴定的候选DHHC，我们鉴定了DHHC 2、5、8、15和20参与HA的酰化。如果在人呼吸道A549细胞中用CRISPR/Cas9单独敲除这些DHHC，则HA的棕榈酰化作用降低，病毒滴度被抑制~1log，表明多个DHHC协同工作。我们现在的目标是创建A549细胞，在其中几个DHHC的表达被同时抑制，直到HA‘S酰化和病毒复制被更严重地抑制。我们将分析已鉴定的DHHC-蛋白是否能酰化甲型流感各亚型的HA、乙型流感的HA和丙型流感的HEF，以及其他被囊膜病毒的膜蛋白。病毒颗粒中的乙酰化减少和HA的脂肪酸模式也将通过质谱学进行密切监测。这将允许确定特定的DHHC蛋白是棕榈酸还是硬脂酸盐所特有的。分子模拟将补充实验数据。已发表的DHHC 15和20的空间结构揭示了一个共价结合的脂肪酸埋藏在由四个跨膜区形成的疏水空腔中。空腔的狭窄末端被DHHC蛋白之间的两个可变氨基酸封闭；这两个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蛋白质的疏水空腔；特别是寻找具有短疏水空腔的DHHC，该疏水空腔可以容纳棕榈酸，但不能容纳硬脂酸。此外，最近公布的HA跨膜区的结构可能有助于确定已鉴定的DHHC蛋白中的底物结合部位，以及HA中的哪些特征是由DHHC蛋白识别的。预测将通过DHHC识别的HA中氨基酸的突变以及随后使用反向遗传学和酰化分析进行分析来验证。

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.