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.

Chlan­da, P., Mekhe­dov, E., Waters, H., Sodt, A., Schwartz, C., Nair, V., Blank, P.S., Zim­mer­berg, J. (2017) Palmi­toy­la­tion con­tributes to mem­brane cur­va­ture in Influen­za A virus assem­bly and hemag­glu­tinin-medi­at­ed mem­brane fusion. J. Virol. doi: 10.1128/JVI.00947–17.

Chlan­da, P., Kri­jnse Lock­er, J., (2017) The sleep­ing beau­ty kissed awake: new meth­ods in elec­tron microscopy to study cel­lu­lar mem­branes. Bio­chem­i­cal Jour­nal, DOI: 10.1042/BCJ20160990

Quemin, E.R.J.*, Chlan­da, P.*, Sachse, M., Forterre, P., Prangishvili, D. and Krupovic, M., (2016) Eukary­ot­ic-like virus bud­ding in Archaea, MBio, doi: 10.1128/mBio.01439–16.*Equal contribution

Chlan­da, P., Mekhe­dov, E., Waters, H., Schwartz, C.L., Fis­ch­er, E.R., Ryham, R.R., Cohen, F.S., Blank, P.S. and Zim­mer­berg, J. (2016), The hemi­fu­sion struc­ture induced by Influen­za virus hemag­glu­tinin is deter­mined by phys­i­cal prop­er­ties of the tar­get mem­branes. Nature Micro­bi­ol­o­gy, doi:10.1038/nmicrobiol.2016.50

Chlan­da, P., Zim­mer­berg, J. (2016), Pro­tein-lipid inter­ac­tions crit­i­cal to repli­ca­tion of the influen­za A virus dur­ing infec­tion. FEBS Lett., doi: 10.1002/1873–3468.12118

Chlan­da, P., Schraidt, O., Kum­mer, S., Rich­es, J., Ober­win­kler, H., Prinz, S., Kräus­slich HG and Brig­gs, J.A.G. (2015), Struc­tur­al analy­sis of the con­tri­bu­tions of indi­vid­ual pro­teins to influen­za assem­bly and mor­phol­o­gy. J Virol, 89(17):8957–66.

Chlan­da, P., Sachse, M. (2014), Cryo-Elec­tron Microscopy of Vit­re­ous Sec­tions. Meth­ods Mol Biol, 1117:193–214.

Kri­jnse Lock­er, J., Chlan­da, P., Sach­sen­heimer, T., Brüg­ger, B. (2013), Poxvirus mem­brane bio­gen­e­sis: rup­ture not dis­rup­tion. Cell Micro­bi­ol, 15(2):190–9.

Chlan­da, P., Car­ba­jal, M.A., Cyrk­laff, M., Grif­fiths, G., Kri­jnse-Lock­er, J. Mem­brane rup­ture gen­er­ates sin­gle open mem­brane sheets dur­ing vac­cinia virus assem­bly. (2009), Cell Host Microbe, 6(1):81–90.

Cyrk­laff, M., Linaroud­is, A., Boicu, M., Chlan­da, P., Baumeis­ter, W., Grif­fiths, G., Kri­jnse-Lock­er, J. (2007), Whole cell cryo-elec­tron tomog­ra­phy reveals dis­tinct dis­as­sem­bly inter­me­di­ates of vac­cinia virus. PLoS ONE, 2(5):e420.