Characterization of Ebola Virus VP30 Phosphorylation with a Phosphospecific Antibody

Ebola virus is a nonsegmented negative-strand RNA virus of the family Filoviridae. Ebola virus is highly pathogenic and classified as a BSL-4 agent. In humans, the virus causes a severe, often fatal disease. Replication and transcription of the viral genome are achieved by viral proteins of the nuc...

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Bibliographic Details
Main Author: Lier, Clemens
Contributors: Becker, Stephan (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2019
Medizin
Subjects:
RNA
RNS
NP
Online Access:PDF Full Text
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Summary:Ebola virus is a nonsegmented negative-strand RNA virus of the family Filoviridae. Ebola virus is highly pathogenic and classified as a BSL-4 agent. In humans, the virus causes a severe, often fatal disease. Replication and transcription of the viral genome are achieved by viral proteins of the nucleocapsid complex, which consists of the viral RNA genome and the viral proteins NP, VP24, L, VP35, and VP30. For replication of the viral genome, only NP, VP35, and L are needed, whereas transcription of individual genes also requires a functional VP30. Previous studies indicated that extensive serine phosphorylation of VP30 impairs viral transcription. Here, we demonstrated that phosphorylation of two VP30 serine residues, namely serine 29 and 31, is both necessary and sufficient for downregulation of VP30's transcriptional support activity. Phosphorylation of VP30 also dynamically modulates the interaction with other viral proteins. For primary transcription immediately after infection of new cells, a phosphorylatable VP30 is a prerequisite. We were able to show that VP30 phosphorylation is essential at early time points of infection to ensure transport of VP30 with the incoming nucleocapsids to the site of primary viral transcription. With the help of a phosphospecific peptide VP30 antibody directed against serine 29 phosphorylation, we further demonstrated that the majority of VP30 is dephosphorylated at position 29 during infection with recombinant Ebola virus. By recombinantly expressing different combinations of viral proteins, we could show that other viral proteins, especially the nucleoprotein NP, decisively influence VP30 phosphorylation. We gathered first evidence showing that VP30 is a substrate of phosphatases recruited by NP into spatial proximity of VP30. Furthermore, we demonstrated that VP30 directly interacts with a so far unknown cellular kinase, which recognizes a common R-X-X-S phosphorylation motif for VP30 serine residue 29. On the basis of these interactions, both VP30-specific phosphatases and kinases are recruited to perinuclear viral inclusion bodies, where they modulate viral transcription and replication.
Physical Description:148 Pages
DOI:https://doi.org/10.17192/z2019.0292