Membrane Biology of Viral Infection
Dr. Petr Chlanda
We are interested in studying how viruses interact with cellular organelles and how they remodeled cells to replicate. Many enveloped viruses such as influenza, SARS-CoV-2 or Ebola must cross the cell membrane during entry and exit. To do so, viruses developed or hijacked different protein machinery, which is able to remodel, fuse or cut membranes in processes dependent on specialized lipids. We use and further develop cryo-electron microscopy (cryo-EM) techniques such as cryo-focused ion beam milling (cryo-FIB), in situ cryo-electron tomography (cryo-ET) and cryo-correlative light and electron microscopy (cryo-CLEM) in conjunction with other imaging methods as fluorescence microscopy and imaging mass spectrometry to solve the puzzle of viral-membrane interactions.
“On a molecular level, virus-infected cell is a vast unexplored universe whose understanding will not be possible without constant effort to advance imaging technologies.”
Research Strategy
We aim to understand the molecular mechanisms underlying the membrane and cellular remodelling processes that are pivotal in the viral infection of RNA membrane-enveloped viruses. Influenza A virus, SARS-CoV-2, or the Ebola virus remodel cells to replicate and evade the immune system and they must cross the cell membrane during entry and exit. Viruses have developed or hijacked different protein machinery, which is able to remodel, fuse, or cut membranes in processes dependent on specialised lipids. We use and further develop cryo-electron microscopy (cryo-EM) techniques such as cryo-focused ion beam milling (cryo-FIB), in situ cryo-electron tomography (cryo-ET), and cryo-correlative light and electron microscopy (cryo-CLEM) in conjunction with other imaging methods, such as fluorescence microscopy and imaging mass spectrometry, to solve the puzzle of viral-membrane interactions.
In particular, we study membrane fusion and entry of spherical and filamentous influenza A viruses and we are interested in how influenza A viruses incorporate segmented genomes into the budding viruses. We study replication organelles and assembly of SARS-CoV-2 and Ebola viruses.
Dr. Petr Chlanda
- +49 (0)6221 54-51231
- petr.chlanda@bioquant.uni-heidelberg.de
Administration
Anna Michalczyk-Schneider
- +49 (0)6221 56-5003
- anna.michalczyk-schneider@med.uni-heidelberg.de
Structural analysis of Ebola virus entry and inclusion bodies
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 the Niemann-pick-C1 receptor (NPC1). We study viral fusion and disassembly and the formation inclusion bodies using Ebola transcription and replication-competent virus-like particles (BSL1) using cryo-CLEM, cryo-FIB/SEM and cryo-ET.
Structural analysis of SARS-CoV-2 assembly
Coronavirus assembly occurs on the membrane of ERGIC cisternae. While S spike protein does not play a role in the formation of new virions, two small transmembrane proteins M and E are responsible for modulating the curvature of the ERGIC membrane and for incorporation of the viral ribonucleoprotein complexes carrying the viral genome. We apply a variety of electron microscopy techniques to characterize the role of individual structural proteins such as M and E proteins in SARS-CoV-2 virus-like particle assembly and release.
Influenza A virus entry and replication
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. We study viral fusion and disassembly in the cells close to their native state using cryo-CLEM, cryo-FIB/SEM and cryo-ET to investigate the role of host factors such as IFITM3 proteins and lipids such as cholesterol on the membrane fusion and virion disassembly. In addition, we study the sorting and trafficking of viral ribonucleoproteins to virion budding sites.
Architecture of pulmonary lamellar bodies
Lamellar bodies (LBs) are lysosome-derived organelles found in alveolar type 2 epithelial cells (AEC2) in the pulmonary alveoli that are at the ends of terminal bronchioles. LB functions as a surfactant factory, it accumulates a large number of phospholipids in the lamellated core composed of tightly packed bilayers. Upon secretion into the alveolar cavity, LB disassembles into a highly organized network called the surfactant layer which reduces the surface tension at the air-water interface to facilitate gas exchange during respiration. Pulmonary surfactants are primary players in the pulmonary immune response against respiratory viruses such as influenza A virus or coronaviruses.
Secondary Ion Mass Spectroscopy imaging of infected cells
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 plan to apply SIMS to vitreous cryo-lamellae 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 will study lipids such as cholesterol, sphingolipids and phosphatidylinositols, which are crucial in host-pathogen interactions as well as in membrane trafficking.
Selected Publications
SARS-CoV-2 nsp3 and nsp4 are minimal constituents of a pore spanning replication organelle.
Zimmermann L, Zhao X, Makroczyova J, Wachsmuth-Melm M, Prasad V, Hensel Z, Bartenschlager R, Chlanda P.
Nat Commun. 2023 Nov 30;14(1):7894. doi: 10.1038/s41467-023-43666-5.
The Ebola virus VP40 matrix layer undergoes endosomal disassembly essential for membrane fusion.
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.
EMBO J. 2023 Jun 1;42(11):e113578. doi: 10.15252/embj.2023113578.
IFITM3 blocks influenza virus entry by sorting lipids and stabilizing hemifusion.
Klein S, Golani G, Lolicato F, Lahr C, Beyer D, Herrmann A, Wachsmuth-Melm M, Reddmann N, Brecht R, Hosseinzadeh M, Kolovou A, Makroczyova J, Peterl S, Schorb M, Schwab Y, Brügger B, Nickel W, Schwarz US, Chlanda P.
Cell Host Microbe. 2023 Apr 12;31(4):616-633.e20. doi: 10.1016/j.chom.2023.03.005.
SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography.
Klein S, Cortese M, Winter SL, Wachsmuth-Melm M, Neufeldt CJ, Cerikan B, Stanifer ML, Boulant S, Bartenschlager R, Chlanda P.
Nat Commun. 2020 Nov 18;11(1):5885. doi: 10.1038/s41467-020-19619-7.
Full publication list of the group leader on PubMed