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.

 


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.

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.

 

 

 

 

 

 

Contact: E-Mail (Last update: 20/11/2023)