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

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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.