Publikationsdatenbank

Structural, enzymological and functional studies on the binding of fatty acids to the haemagglutinin and the M2 membrane protein of the influenza virus (2023)

Art

Hochschulschrift

Autor

Meng, Xiaorong (WE 5)

Quelle

Berlin, 2023 — VII, 99 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/40992

Kontakt

Institut für Virologie

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51833
virologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Influenza viruses are major respiratory viruses, that cause annual epidemics and occasional pandemics. S-palmitoylation, the covalent attachment of long chain fatty acids to membrane proteins, is catalyzed by members of the DHHC family of protein acyl transferases. Protein palmitoylation regulates cellular processes and pathogens like Influenza viruses use the S-palmitoylation machinery of host cells to promote infection, but how substrates are recognized by DHHCs is unknown. In the first part of my work, I used the M2 protein of influenza A virus, which has one acylated cysteine at a membrane-near amphiphilic helix (AH), to study molecular determinants for palmitoylation and binding to its cognate enzyme DHHC20. After fusion of the amphiphilic helix of M2 with the fluorescent protein RFP, the reporter protein was membrane bound and palmitoylated. Changing the biophysical properties of the amphiphilic helix, its truncation, or destroying its secondary structure greatly reduced but does not eliminate the S-palmitoylation of full-length M2. Furthermore, the exchange of a kink-inducing glycine in the transmembrane (TM) region increased acylation. DHHC20, one of the enzymes involved in palmitoylation of M2, can be co-precipitated with M2 mutants and two of them bind even more strongly than M2 wildtype. Molecular dynamic simulations, performed by Clark Templeton in the group of Cecilia Clementi (Theoretical and Computational Biophysics, Free University Berlin) revealed that M2 associates via both its TM and AH with DHHC20. Amino acids of the helix interact with the catalytically important DHHC and TTXE motifs of the enzyme and some contacts are lost in M2 mutants. In the second part of my thesis, I studied the role of palmitoylation of the main glycoprotein hemagglutinin of Influenza B virus for virus replication and identified the DHHC enzymes required for the acylation. I found that palmitoylation is essential for influenza B virus replication since removing the two sites where the palmitate is attached to the HA protein or the cysteine located at the end of the cytoplasmic tail prevented generating viable viruses. Viruses with an exchange of the membrane-distal cysteine rapidly reverted back to wild-type viruses. Using Brefeldin A to block the exit of proteins from the endoplasmic reticulum (ER), I show that palmitoylation of HA of Influenza B virus occurs in the ER, whereas acylation of HA of Influenza A virus happens also in the Golgi. Infecting HAP-1 cells where genes for individual DHHCs have been inactivated revealed that HA of the Influenza B virus is acylated by the ER-localized DHHCs 1, 2, 4, and 6, which are mostly different from those previously identified to be involved in palmitoylation of HA of Influenza A virus. Overall, these results prove that palmitoylation of HA plays an essential role in the replication of Influenza B virus, as has previously been demonstrated for Influenza A virus. However, the responsible enzymes and their intracellular localization are different for Influenza A and B viruses. Overall, my research enhances our understanding of the molecular mechanisms underlying influenza virus replication and the role of S-palmitoylation in viral infection. The findings provide valuable insights into the interplay between viral proteins and host cellular processes, offering potential targets for antiviral interventions. Additionally, the identification of virus-specific differences in palmitoylation pathways emphasizes the importance of studying individual influenza strains to develop tailored therapeutic strategies.