The role of acylation of Gp5/M of porcine reproductive and respiratory syndrome virus for assembly and budding of virus particles elucidated by super resolution microscopy.

• Applicants: Dr. Susann Kummer; Privatdozent Dr. Michael Veit
• Subject Area: Veterinary Medical Science
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 525482931

Porcine respiratory and reproductive syndrome virus (PRRSV) is the most important pathogen in swine herds, but its membrane proteins and essential aspects of virus replication, such as virus budding, are poorly studied. In a previous DFG-project, we showed that the most abundant membrane protein of PRRSV, the Gp5/M dimer, is acylated at cysteines located at the end of the transmembrane region of Gp5 and M. The modification is essential for virus replication, especially for the assembly and release of virus particles. The enzymes that acylate Gp5/M have not been identified yet, but transfer of fatty acids to proteins is mediated by one or several of the 23 members of the DHHC family of proteins. We also used the artificial intelligence-based system of alphafold2 to predict a reliable structure of Gp5/M. It revealed six curved and tilted transmembrane helices, three from each protein. The third transmembrane helix extends into the cytoplasm and contains the acylation sites, which are located on the hydrophobic site of an amphiphilic helix. This resembles the amphiphilic helix of M2 of Influenza A virus, that inserts into the membrane to induce curvature. In this project, we aim to test the hypothesis that fatty acids bound to Gp5 and M are required for the clustering of Gp5/M dimers. We will use super-resolution microscopy to analyse whether Gp5/M dimers form oligomers at Golgi membranes and whether clustering is reduced when fatty acid attachment sites are removed from Gp5 and/or M. To achieve this goal, Susan Kummer, a specialist for STED microscopy from the Robert Koch Institute is a co-applicant. Mechanistically, the fatty acids might recruit cholesterol to the viral budding site, as has been recently described for acylation of the spike of SARS-CoV-2. This could produce a nanodomain in the membrane that promotes the assembly of further viral components, as has been described for the assembly of influenza viruses at the plasma membrane. This hypothesis will be tested with a photoactivated cholesterol analogue, which we have already used to identify a cholesterol binding site in the HA of influenza A viruses. We will also investigate the role of the amphiphilic helices of Gp5 and M for virus replication and acylation. Finally, we aim to identify the DHHC enzymes that acylate Gp5 and M. We hypothesise that DHHC 1, 4, 6 and/or 20, the only ER resident DHHCs, are the most likely candidates as Gp5 and M are acylated when expressed alone and not transported beyond the ER. We will use the same methods (siRNA and CRISPR/Cas9-mediated knock-out of DHHC genes) that we recently established to identify the DHHCs required for acylation of HA of Influenza A virus. This project not only provides molecular information for an important step in the replication cycle of Arteriviruses, but also clarifies similarities and differences in virus budding at internal membranes and at the plasma membrane.

• DFG Programme: Research Grants