1 | Influenza A virus entry and membrane fusion

Influen­za A virus (IAV) is a pleiomor­phic, enveloped virus that enters the host cell either by endo­cy­to­sis or macropinocy­to­sis and fus­es with the endo­so­mal mem­brane in a Hemag­glu­tinin (HA)-mediated process that occurs at low pH. Nei­ther the struc­ture of the HA fusion inter­me­di­ates nor the spa­tial ori­en­ta­tion of the post-fusion HA with respect to the fusion pore are known. We use cryo-ET and subto­mo­gram aver­ag­ing to fur­ther char­ac­ter­ize the struc­ture of HA fusion inter­me­di­ates. In addi­tion, we study viral fusion and dis­as­sem­bly in the cells close to their native state using cryo-CLEM, cryo-FIB/SEM and cryo-ET. This will allow to fur­ther inves­ti­gate the role of host fac­tors such as aggre­some machin­ery, cytoskele­ton, IFITMs pro­teins and lipids such as cho­les­terol on the mem­brane fusion and viri­on disassembly.

2 | Structual analysis of Ebola virus entry intermediates in vitro and in situ

Ebo­la virus (EBOV) assem­bles into long fil­a­men­tous viri­ons (1–15 μm) at the plas­ma mem­brane, which upon release, enter epithe­lial cells by macropinocy­to­sis. The neg­a­tive sin­gle strand­ed RNA genome is coiled across a length of ~0.9 μm and pro­tect­ed by the nucle­o­cap­sid com­posed of the nucle­o­pro­tein (NP), VP35, VP40, and VP24 pro­teins. The Ebo­la fusion gly­co­pro­tein (GP) is pro­te­olyt­i­cal­ly processed in the late endo­some by low-pH sen­si­tive cathep­sin pro­teas­es to a 19 kDa frag­ment, which binds to Nie­mann-pick-C1 recep­tor (NPC1). The 19 kDa frag­ment bound to NPC1 togeth­er with yet unknown factor(s) is able to induce mem­brane fusion allow­ing the release of the genome. Both the dis­as­sem­bly of the fil­a­men­tous virus and the mech­a­nism under­ly­ing GP medi­at­ed mem­brane fusion in the endo­somes are poor­ly under­stood process­es and have not been struc­tural­ly char­ac­ter­ized. We use non-infec­tious EBOV VLPs, which are com­posed of five major struc­tur­al pro­teins (GP, NP, VP40, VP35, and VP24) and are struc­tural­ly sim­i­lar to EBOV to study EBOV virus entry inter­me­di­ates by cryo-CLEM, cryo-FIB/SEM and cryo-ET both in vit­ro and in liv­ing cells.

3 | Spatial lipidomics and viral infection

Sec­ondary ion mass spec­trom­e­try (SIMS) allows non-inva­sive imag­ing of chem­i­cal­ly unmod­i­fied lipids with high chem­i­cal speci­fici­ty. How­ev­er, SIMS has so far been only per­formed on chem­i­cal­ly fixed and dehy­drat­ed sam­ples (sam­ple prepa­ra­tion pro­ce­dures known to severe­ly alter mem­brane struc­ture). In col­lab­o­ra­tion with Tom Wirtz (Lux­em­bourg Insti­tute of Sci­ence and Tech­nol­o­gy), we will apply SIMS to vit­re­ous cryo-lamel­las of the cells pre­pared by focused-ion beam milling to pro­vide a spa­tial map of lipids in cel­lu­lar organelles at native con­di­tions. The lipid map will be sub­se­quent­ly cor­re­lat­ed to the mem­brane struc­tures observed by cryo-ET. We study lipids such as cho­les­terol which is cru­cial in host-pathogen inter­ac­tions as well as in mem­brane trafficking.

Win­ter SL, Chlan­da P, (2023) The Art of Viral Mem­brane Fusion and Pen­e­tra­tion, Sub­cell Biochem, Book: Virus Infect­ed cells. doi: 10.1007/978–3‑031–40086-5_4.

Zim­mer­mann L, Zhao X, Makroczy­o­va J, Wachsmuth-Melm W, Prasad V, Hensel Z, Barten­schlager R, Chlan­da P. (2023) SARS-CoV­‑2 nsp3 and nsp4 are min­i­mal con­stituents of a pore span­ning repli­ca­tion organelle. Nature Communications,14(1):7894. doi: 10.1038/s41467-023–43666‑5

Zim­mer­mann L, Chlan­da P. Cryo-elec­tron tomog­ra­phy of viral infec­tion — from appli­ca­tions to biosafe­ty. Curr Opin Virol. 2023 Aug;61:101338. doi: 10.1016/j.coviro.2023.101338.

Win­ter SL, Golani G, Loli­ca­to F, Vall­bracht M, Thiya­gara­jah K, Ahmed SS, Lücht­en­borg C, Fack­ler OT, Brüg­ger B, Hoe­nen T, Nick­el W, Schwarz US, Chlan­da P, (2023) The Ebo­la virus VP40 matrix under­goes endo­so­mal dis­as­sem­bly essen­tial for mem­brane fusion, EMBO J. 42(11):e113578. doi: 10.15252/embj.2023113578.

Klein S, Golani G, Loli­ca­to F, Bey­er D, Her­rmann A, Wachsmuth-Melm M, Red­dmann N, Brecht R, Lahr C, Hos­sein­zadeh M, Kolovou A, Schorb M, Schwab Y, Brüg­ger B, Nick­el W, Schwarz US, Chlan­da P, (2023) IFITM3 blocks viral entry by sort­ing lipids and sta­bi­liz­ing hemi­fu­sion, Cell Host&Microbe, S1931-3128(23)0019–9, doi: 10.1016/j.chom.2023.03.005.

Win­ter SL, Chlan­da P, (2021) Dual-axis Vol­ta phase plate cryo-elec­tron tomog­ra­phy of Ebo­la virus-like par­ti­cles reveals actin-VP40 inter­ac­tions, J Struct Biol, 107742. doi: 10.1016/j.jsb.2021.107742.

Klein S, Wim­mer WH, Win­ter SL, Kolovou A, Lake­ta V, Chlan­da P, (2021) Post-cor­re­la­tion on-lamel­la cryo-CLEM reveals the mem­brane archi­tec­ture of lamel­lar bod­ies. Com­mu­ni­ca­tions Biol­o­gy, 2021 Jan 29;4(1):137. doi: 10.1038/s42003-020–01567‑z.

Klein S, Wachsmuth-Melm M, Win­ter SL, Kolovou A, Chlan­da P. (2021) Cryo-cor­rel­a­tive light and elec­tron microscopy work­flow for cryo-focused ion beam milled adher­ent cells. Meth­ods Mol Biol. Cor­rel­a­tive Light and Elec­tron Microscopy, IV Vol­ume 162, Chap­ter 12.

Klein S, Cortese M, Win­ter SL, Wachsmuth-Melm M, Neufeldt CJ, Cerikan B, Stan­i­fer ML, Boulant S, Barten­schlager R, Chlan­da P. (2020) SARS-CoV­‑2 struc­ture and repli­ca­tion char­ac­ter­ized by in situ cryo-elec­tron tomog­ra­phy. Nature Com­mu­ni­ca­tions, 11, 5885 doi.org/10.1038/s41467-020–19619‑7.

Klein S, Müller TG, Khalid D, Son­ntag-Buck V, Heuser AM, Glass B, Meur­er M, Morales I, Schillak A, Freis­taedter A, Ambiel I, Win­ter SL, Zim­mer­mann L, Nau­mos­ka T, Bubeck F, Kir­rmaier D, Ull­rich S, Bar­reto Miran­da I, Anders S, Grimm D, Schnit­zler P, Knop M, Kräus­slich HG, Dao Thi VL, Börn­er K, Chlan­da P. (2020), SARS-CoV­‑2 RNA Extrac­tion Using Mag­net­ic Beads for Rapid Large-Scale Test­ing by RT-qPCR and RT-LAMP. Virus­es. 12(8):863. doi: 10.3390/v12080863.