S-Acylation of hemmagglutinin of influenza virus with different fatty acids - structural requirements and functional consequences

流感病毒的血凝素（HA）在位于跨膜区（TMR）的内叶或胞质尾区（CT）中的三个半胱氨酸处被S-酰化。HA的酰化是病毒复制所必需的，影响病毒通过膜融合和/或病毒颗粒从感染细胞出芽进入细胞。在之前柏林（Veit）和莫斯科（Kordyukova）之间的合作中，我们使用质谱和MS/MS测序显示位于HA TMR中的半胱氨酸仅含有硬脂酸盐，而CT中的两个半胱氨酸被棕榈酸盐修饰。通过这个联合项目提案，我们试图确定差异酰化的结构基础，并探索脂肪酸类型对HA功能的重要性。Efremov教授是计算机建模（分子动力学和蒙特卡罗模拟）专家，他将加入该项目，以确定HA（和其他病毒酰基蛋白）的TMR和CT的构象，以识别棕榈酰化和硬脂酰化半胱氨酸残基周围存在的特殊信号。预测将通过HA的诱变、表达和脂肪酸分析进行测试。最后，使用三种已经可用的重组流感病毒，每种都具有HA，其中一个脂肪酸结合位点缺失（因此其硬脂酸酯含量存在很大差异），我们将分析S-酰化是否影响HA与基质蛋白M1的结合，这是感染性病毒颗粒出芽的先决条件。

The hemagglutinin (HA) of influenza virus is S-acylated at three cysteines located either in the inner leaflet of the transmembrane region (TMR) or in the cytoplasmic tail (CT). Acylation of HA is essential for virus replication, affecting cell entry of viruses via membrane fusion and/or budding of virus particles from the infected cell. In a previous collaboration between Berlin (Veit) and Moscow (Kordyukova) we used mass-spectrometry and MS/MS sequencing to show that the cysteine located in the TMR of HA contains only stearate whereas two cysteines in the CT are modified with palmitate. With this joint project proposal we attempt to identify the structural basis for differential acylation and explore the significance of the type of fatty acids for the function of HA. Prof. Efremov, a specialist for computer modelling (molecular dynamics and Monte Carlo simulations), will join the project to determine the conformation of the TMR and CT of HA (and of other viral acylproteins) to identify peculiar signals present around palmitoylated and stearoylated cysteine residues. The predictions will be tested by mutagenesis, expression and fatty acid analysis of HA. Finally, using three already available recombinant influenza viruses, each having HA with one fatty acid binding site deleted (and as a consequence large differences in their stearate content), we will analyze whether S-acylation affects binding of HA to the matrixprotein M1, which is a prerequisite for the budding of infectious virus particles.