DynamicMembrane - Understanding mechanisms of membrane rupture and repair
Cell Biology
Final Report Abstract
The membrane-enveloped vaccinia virus follows an unusual mode of membrane recruitment with the membrane biogenesis being initiated by a preassembling viral protein scaffold. The scaffold is thought to recruit small membrane structures that rupture and form short half-moon shaped membranes which eventually grow into a membrane sphere enclosing the viral core proteins and forming the immature virus. How viral structural membrane proteins and viral membrane assembly proteins operate in an orchestrated mode to initiate this process is not well understood. The aim of the project was to understand the role of both lipids and a small viral protein, the gene product of A11, in the unconventional membrane biogenesis of VACV. In preliminary work we could show that the membrane of purified VACV is enriched in several lipid species when compared to HeLa cells used to grow the virus. These concerned in particular negatively charged lipids such as phosphatidic acid and phosphoinositol-containing glycerolipids. We hypothesized that membrane rupture and subsequent membrane formation is mediated by an interplay between cellular membrane lipids and viral proteins. To test this hypothesis, we had suggested a combination of in vitro and cellular studies, specifically to investigate the contribution of the viral membrane assembly protein A11 to this process. We identified a C- terminally located amphipathic helix suggesting to serve as an in plane membrane (IPM) anchor. To test whether this IPM anchor contributes to membrane remodeling events during the viral assembly process, a number of A11 constructs were expressed and purified. However, we could not assign strong membrane binding to the isolated domain of IPM anchor and the domain also did not show membrane remodeling activity. Studies investigating the impact of the full-length protein and of additional domains on membrane remodeling are still ongoing, and include also other viral membrane assembly proteins, in particular H7. In cellular studies H7 was found to play an unexpected role in vaccinia virus assembly, in particular in the formation of D13 hexagons and high-order honey-combs-like structures that are in the center of scaffold formation during the early steps of viral assembly. Accompanying in vitro experiments studying H7 and D13 in a liposomal system are still ongoing, as are cellular studies combined with proteomics and lipidomics to investigate the interplay of viral proteins and host lipids in vaccinia virus assembly.
Publications
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2017. The host-cell restriction factor SERINC5 restricts HIV-1 infectivity without altering the lipid composition and organization of viral particles. J Biol Chem 292:13702-13713
Trautz B, Wiedemann H, Lüchtenborg C, Pierini V, Kranich J, Glass B, Kräusslich HG, Brocker T, Pizzato M, Ruggieri A, Brügger B, Fackler OT
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2018. Entry and Disassembly of Large DNA Viruses: Electron Microscopy Leads the Way. J Mol Biol 430:1714-1724
Quemin ER, Corroyer-Dulmont S, Krijnse-Locker J
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2019. Quantification of phosphoinositides reveals strong enrichment of PIP2 in HIV-1 compared to producer cell membranes. Sci Rep 9:17661
Mücksch F, Citir M, Lüchtenborg C, Glass B, Traynor-Kaplan A, Schultz C, Brügger B, Kräusslich HG
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CerS6-Derived Sphingolipids Interact with Mff and Promote Mitochondrial Fragmentation in Obesity. 2019. Cell 177:1536-1552
Hammerschmidt P, Ostkotte D, Nolte H, Gerl MJ, Jais A, Brunner HL, Sprenger HG, Awazawa M, Nicholls HT, Turpin-Nolan SM, Langer T, Krüger M, Brügger B, Brüning JC
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Protease-resistant streptavidin for interaction proteomics. 2020. Mol Syst Biol 16:e9370
Rafiee MR, Sigismondo G, Kalxdorf M, Förster L, Brügger B, Béthune J, Krijgsveld J
