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

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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.