DynamicMembrane - Understanding mechanisms of membrane rupture and repair

• Applicant: Professorin Dr. Britta Brügger
• Subject Area: Virology ; Cell Biology
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 316659730

Within eukaryotic cells membrane compartments are closed entities that communicate by vesicular transport, controlled by specific protein machineries. Being obligatory intracellular parasites, viruses acquire their membrane from the host, in a process mimicking the formation of vesicles. Nucleo-cytoplasmic large DNA viruses (NCLDVs), a family of large DNA viruses, are an exception to this rule and follow an unconventional pathway of membrane acquisition. During infection they form open membrane intermediates, likely the result of membrane rupture, from which they build an open membrane sphere. The latter is shaped by the viral scaffold protein, a protein conserved among most NCLDVs. The membrane sphere remains open until the viral genome has been taken up, upon which the membrane closes and the particle matures into an infectious virion. We proposed that this unusual membrane assembly is controlled by a machinery, common to NCLDVs, which includes both (viral) proteins and lipids. Together they mediate membrane rupture, stabilization of open membrane ends, the formation of the open sphere and its subsequent closure after DNA-uptake. Our recent lipid mass spectrometry (MS) results of purified vaccinia virus (VACV, member of the NCLDVs) showed that its envelope is enriched in lipid species that have been implicated in membrane rupture/destabilization. Moreover, preliminary data obtained by 3D-electron tomography (ET) identified a VACV protein, required for membrane rupture in infected cells. Within the frame of this proposal we will search for interacting partners of this VACV protein during infection. The VACV-protein will be expressed and purified with or without its interacting partners and its structure analyzed by X-ray crystallography as well as by cryo-EM. For the former we will rely on the collaboration with Dr. Coulibaly (Monash, Melbourne), an expert on X-ray crystallography of viral proteins, in particular of VACV proteins. In parallel we will continue lipid-analyses and identify minor lipid species and specific lipid classes of purified VACV. We propose to complement these data by lipid-MS of open VACV membranes, isolated and affinity-purified from infected cells. The role of specific lipids in membrane rupture will be analyzed in vitro as well as in vivo. In vitro the VACV protein required for rupture will be reconstituted in artificial membranes (liposomes) containing lipids identify by MS to be enriched in the viral membrane. Its effect on liposomes will be analyzed by content release and cryo-ET. In vivo we aim at inhibiting the synthesis of specific lipids in infected cells and assess effects on VACV assembly by ET. Our study sheds light on mechanisms of membrane rupture and repair that may find applications in targeted delivery of compounds in cells. Evolutionary, it may shed light on a mechanism that is either rare in modern eukaryotes or was present in an early eukaryote/archaea/bacteria but was lost during evolution.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partner: Professorin Jacomine Krijnse Locker, Ph.D.