title: Analysis of protein-sphingolipid interactions in Influenza A-infected cells
creator: Förster, Laura-Christin
subject: ddc-570
subject: 570 Life sciences
description: Influenza A virus (IAV) is a human respiratory pathogen causing seasonal en-demic and irregular pandemic flu infections. The ability to undergo rapid anti-genic changes is a reason for the persistent clinical relevance of IAV as contin-uously novel subtypes emerge, severely compromising vaccine effectiveness.  IAV budding is initiated by the viral glycoproteins hemagglutinin (HA) and neu-raminidase (NA) and occurs at apical plasma membrane regions, referred to as microdomains or membrane rafts, which are enriched in cholesterol and (gly-co)sphingolipids. Sphingolipids (SLs) derive from the amino alcohol sphingo-sine and represent major building blocks of cellular membranes and important cell signaling molecules. SLs seem to play a crucial role in viral assembly as a defect in sphingomyelin (SM) biosynthesis has been shown to compromise cell surface transport of HA and NA, as well as budding and release of progeny viri-ons. In addition, SLs are significantly enriched in the viral envelope. However, direct evidence for an IAV-triggered modulation of SL metabolism and the con-tribution of specific SL classes, species, metabolites or SL metabolising en-zymes to viral propagation is still lacking.  The aim of this thesis was to shed light on the role of SLs and SL-binding host proteins in the IAV replication cycle. Photoactivatable and clickable sphingosine (pacSph) was employed to monitor protein-SL interactions in sphingosine-1-phosphate lyase (S1PL)-deficient cells. Here, the goal was i) to analyse the SL-binding potential of viral proteins, especially HA and NA, of different IAV sub-types, ii) to perform a proteome-wide mapping of protein-sphingolipid interac-tions at early and late stages of infection, and iii) to study roles of long-chain SL species for IAV propagation using CRISPR/Cas9-mediated ceramide synthase 2 (CerS2) knock-out (KO) cells.   Transfection and infection experiments suggest that HA and NA homologues display different SL-binding efficiencies. Employing a SILAC-based proteomic approach, a set of proteins was identified showing altered SL-interaction dy-namics in infected cells. Here, integrin beta-1 (ITGB1) was further investigated as a potential pro-viral SL-binding host target. Knock-out cell models showed that loss of CerS2 resulted in increased infection efficiency in HeLa cells. Nota-bly, the KO of CerS2 correlated with an elevation of dihydro-SL (DHSL) levels. DHSLs have been reported to increase upon IAV and other viral infections, and might thus also be responsible for the observed pro-viral effect. How the ab-sence of CerS2 affects cellular DHSL levels and how this alters host cell infec-tivity remains to be studied in future experiments.   Together these data provide novel insights into the role of sphingolipids in the IAV replication cycle and might eventually paving the path for new therapeutic targets in the combat against IAV.
date: 2019
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/26959/1/Laura_PhDthesis_PDF-A.pdf
identifier: DOI:10.11588/heidok.00026959
identifier: urn:nbn:de:bsz:16-heidok-269599
identifier:   Förster, Laura-Christin  (2019) Analysis of protein-sphingolipid interactions in Influenza A-infected cells.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/26959/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng