Summary Project description 2nd funding period
Type A influenza viruses (IAV) are the main cause of influenza, a severe respiratory illness. IAV buds from the apical surface of polarized epithelial cells, and the viral membrane has a raft-like lipid composition. Different IAV strains exhibit spherical or filamentous morphology with clinical specimens mostly being filamentous. The viral morphotype depends on the IAV M1 matrix protein and the host cell. IAV enters its target cell mostly via clathrin-mediated endocytosis, but also by macropinocytosis. This is followed by fusion from acidic endosomes mediated by the viral hemagglutinin (HA) protein, and nuclear trafficking of the genomic RNA segments bound to viral nucleoprotein (NP). Several host factors, including proteins and membrane sphingolipids, have been implicated in supporting and restricting IAV infection, but their mechanisms of action are only partly known.
This project combines the expertise of the Kräusslich group in virology and super-resolution and correlative light and electron microscopy (CLEM) with that of the Brügger group regarding functionalized lipids and lipidomics to gain a better understanding on protein-lipid interactions in IAV replication and spread. In the current funding period, we established infection systems for different IAV strains (H1N1 and H3N2, spherical and filamentous) in cell lines and primary human cells, and devised and validated labeling strategies for the virus. In addition, we studied early IAV replication including recruitment of the host factor IFITM3 by confocal and super-resolution microscopy and performed an unbiased screen to identify sphingolipid-binding Proteins relevant for IAV infection. We collaborated with Briggs on structural analysis of M1 and HA proteins inside IAV particles. In the next funding period, we will focus on IAV transfer from the producing to the target cell.
We plan to study the functional relevance of protein-sphingolipid interactions in IAV infection and the entry pathway and recruitment of host cell factors for filamentous versus spheroidal IAV. Specifically, we will concentrate on imaging cell-to-cell transfer of spheroidal and filamentous IAV using microscopy of fixed and live cells and including CLEM; this analysis will extend to host factors and lipids proposed to be involved in virus entry.
Furthermore, we will build on our identification of changes in host protein-sphingolipid interactions and determine on a molecular level their functional relevance for IAV infection. Finally, we will expand our infection systems to 3D lung organoids of murine and human origin (with Boulant, Dao Thi and external collaborators).
This will be complemented by collaborations on structural analyses of IAV with Chlanda and Briggs and on physico-chemical parameters of the endocytic cell-virus machinery at the nanoscale with Cavalcanti-Adam/Spatz.