Identifizierung und Charakterisierung des zellulären Interaktoms des Lassavirus Matrixproteins Z
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Philipps-Universität Marburg
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Highly pathogenic Lassa virus (LASV) poses a major public health threat in West Africa. Protein-protein interactions (PPI) between viral and host proteins play an important role in various stages of the viral life cycle, including virus entry, virus replication, and virus release. In addition, these PPIs can also modulate cellular signaling pathways and biological processes during infection. The LASV matrix protein Z has important structural and functional roles in the viral life cycle, such as regulating viral RNA synthesis, orchestrating viral assembly and release, and inhibiting the interferon-mediated immune response. However, our understanding of the molecular mechanisms and host-protein interactions by which the Z protein exerts its diverse functions is still limited. In the present dissertation project, proximity-dependent biotin identification 2 (BioID2) was used for affinity purification in combination with mass spectrometric analysis for the detection and identification of PPI networks of the LASV Z protein in living cells. Stringent statistical filtering revealed 584 potential host interaction candidates of Z protein.
One of the identified host factors was the septin regulator Cdc42EP1, an effector protein of the Rho GTPase Cdc42. Functional validation using co-immunoprecipitation (co-IP) analyses and confocal microscopy confirmed a physical interaction and subcellular co-localization between LASV Z and Cdc42EP1. Further molecular characterization demonstrated that the interaction between the Z protein and Cdc42EP1 depends on the Cdc42 and Rac interactive binding (CRIB) domain of Cdc42EP1, which normally mediates binding to Cdc42. Interestingly, recombinant expression of Cdc42EP1 regulates the release of Z-induced virus-like particles (VLPs) by modulating septin filaments. Infection studies further showed that LASV induces rearrangement of the cellular septin cytoskeleton. Pharmacological inhibition of septin polymerization with the drug forchlorfenuron resulted in impaired plasma membrane transport of Z protein and reduced release of VLPs and authentic LASV. This underscores the importance of a dynamic septin network during LASV infection. Septins regulate the microtubule as well as the actin cytoskeleton, which also play an important role in the LASV replication cycle. Whereas the microtubule network is involved in plasma membrane transport of the Z protein, a dynamic actin cytoskeleton appears to be required in virus entry and release at the plasma membrane. This work revealed cell-to-cell virus spread via actin-rich filopodia as a novel release mechanism of LASV. In summary, Cdc42EP1 was identified as an important host factor regulating the release of Z-induced VLPs and infectious LASV particles.
The cellular mTOR pathway was one of the most enriched signaling pathways in the LASV Z interactome. For example, the ragulator subunit LAMTOR1 was identified as a putative interaction partner of the Z protein in the interactome analyses. The major function of the ragulator complex is lysosomal scaffolding for amino acid-dependent mTORC1 recruitment and activation. Using co-IP analyses and co-localization studies, the ragulator complex was confirmed as a physical interaction partner of the Z protein. Functional validations indicated that LAMTOR1 is an antiviral factor in Z-mediated VLP release. The possible mechanism suggests the role of LAMTOR1 in the intracellular positioning of late endosomes and lysosomes. In contrast, its function in mTORC1 activation does not appear to play a role in the antiviral activity against Z. The lassaviral surface glycoprotein GP, matrix protein Z, and nucleoprotein NP, as a marker for ribonucleoprotein complexes, co-localized with late endosomes at the plasma membrane in infected cells. The importance of late endosomes and exosome biogenesis for LASV release was also confirmed by electron microscopic characterization of LASV Z-positive extracellular vesicles and LASV-infected cells. Together, this doctoral project revealed previously unknown release mechanisms of LASV. A more comprehensive knowledge of virus-host interactions could open new possibilities for the development of treatment options against the highly pathogenic LASV.
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Created: 2024Issued: 2024-10-30Updated: 2024-10-30
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Medizin
Publisher
Philipps-Universität Marburg
Language
ger
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DoctoralThesis
Keywords
interactomeVirusreplikationmatrix proteinMatrixproteinVirologievirus replicationmTORmTORLassavirusZytoskelettBioID2virologyLAMTOR1Interaktomvirus releaseVirusfreisetzungCytoskeletonCdc42EP1Lassa virus
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570
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Ehlert, Birthe: Identifizierung und Charakterisierung des zellulären Interaktoms des Lassavirus Matrixproteins Z. : Philipps-Universität Marburg 2024-10-30. DOI: https://doi.org/10.17192/z2024.0152.
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This item has been published with the following license: In Copyright