title: Regulation of Ebola virus transcription by the viral nucleoprotein: Impact of protein interactions and post-translational modifications
title: Regulierung der Ebola-Virus-Transkription durch das virale Nukleoprotein: Auswirkungen von Proteininteraktionen und posttranslationalen Modifikationen
titleType: AlternativeTitle
Creators: name: Kämper, Lennart
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: post-translational modification
Nucleoprotein
Medical sciences Medicine
Medizin
Ebola virus

description: The Ebola virus (EBOV) belongs to the family Filoviridae and the order Mononegavirales. Sporadic EBOV outbreaks in sub–Saharan Africa are characterized by a high lethality rate of 40-90%. Replication and transcription of the negative stranded viral genome are facilitated by the viral polymerase L and its co-factor the viral protein (VP) 35. While these two proteins are sufficient to enable viral replication, transcription is additionally dependent on the transcription factor VP30. Whether VP30 is part of the transcriptase complex is dependent on its phosphorylation state: dephosphorylated VP30 actively supports viral transcription, in contrast, the phosphorylated form is no longer recruited by the polymerase complex and thus transcription is no longer supported. The cellular protein phosphatase 2A (PP2A) was identified to play a major role in VP30 dephosphorylation and is therefore essential for viral transcription. The viral nucleoprotein (NP) plays a key role for VP30 dephosphorylation by recruiting both proteins, which brings them in close contact to each other enabling VP30 dephosphorylation. NP is a highly versatile protein that is not only essential for the encapsidation of genomic and anti-genomic RNA but has likewise an important role in recruiting viral as well as host proteins to facilitated viral transcription and replication. 
In the first part of this study, the influence of NP on efficient VP30 dephosphorylation through PP2A was investigated. We could show that both proteins require site-specific recruitment by NP, which brings them in close contact. Further separation of the binding sites on NP led to a reduction in primary transcription that could be linked to an increased VP30 phosphorylation state. This phenotype was further confirmed in rec. EBOV rescue systems, showing an attenuation of a recombinant virus with an increased distance between the PP2A and VP30 binding sites on NP. In this process, we identified a compensatory point mutation that emerged in the NP gene during one of the rescue attempts (NP T603I). Following experiments showed that the NP mutation completely restored viral propagation of an otherwise non-viable virus. The mutation was investigated individually as well as in context of the NP linker mutants in order to characterize its drastic effect on viral propagation. This revealed a specific compensation of the linker related growth defect by NP T603I while the mutation on its own was detrimental for the viral life cycle. Through mass spectrometry analysis, we were able to show phosphorylation of NP T603 in viral particles whereas phosphorylation could not be detected after cellular expression. By using phosphomimetic NP T603 mutants we could show that dynamic phosphorylation at this position within the same NP molecule is important for efficient viral propagation, which is the first evidence for a functional importance of NP phosphorylation. 
Besides PP2A and VP30, EBOV NP is also known to interact with the lysine methyltransferase SET and MYND domain-containing protein 3 (SMYD3). Interestingly its interaction site is located between the binding sites of PP2A and VP30 and a regulative role on transcription has been proposed. However, our results did not suggest an important role of SMYD3 or its enzymatical function for the viral life cycle. Excess amounts of SMYD3 expression led to an inhibition of EBOV transcription and replication, while it had no effect on MARV and LLOV life cycle modeling systems. Despite SMYD3 likely being dispensable for the EBOV life cycle, we could, for the first time, detect methylation of NP at several arginine and lysine residues through mass spectrometry. Incorporation of different members of the protein arginine methyltransferase (PRMT) family into viral particles suggests their involvement in EBOV NP methylation, which is an interesting finding that will open up new research possibilities. 
This study emphasizes the essential role of NP as a regulator of viral transcription and replication. NP facilitates many different functions during the viral life cycle, which is regulated via the interaction with viral and cellular proteins as well as RNA. These processes are often influenced by post translational modifications. Our study identifies the large intrinsically disordered region in the C-terminal part of the protein as an important structural component mediating PP2A, SMYD3, and VP30 binding. The same region is also heavily modified by PTMs and underlies a high evolutionary pressure. This specialized region of NP enables structural variability that is hypothesized to be an important factor to fulfill the large variety of EBOV NP interactions and hence functions during the viral life cycle.