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The nucleoprotein of Marburg virus is target for multiple cellular kinases
B Lötfering1, E Mühlberger, T Tamura
1Institut für Virologie der Philipps-Universität Marburg, Robert-Koch-Strabetae 17, Marburg, D-35037, Germany.
Abstract:
The nucleoprotein (NP) of Marburg virus is phosphorylated at serine and threonine residues in a ratio of 85:15, regardless of whether the protein is isolated from virions or from eukaryotic expression systems. Phosphotyrosine is absent. Although many potential phosphorylation sites are located in the N-terminal half of NP, this part of the protein is not phosphorylated. Analyses of phosphorylation state and phosphoamino acid content of truncated NPs expressed in HeLa cells using the vaccinia virus T7 expression system led to the identification of seven phosphorylated regions (region I*, amino acids 404-432; II*, amino acids 446-472; III*, amino acids 484-511; IV*, amino acids 534-543; V*, amino acid 549; VI*, amino acids 599-604; and VII*, amino acid 619) with a minimum of seven phosphorylated amino acid residues located in the C-terminal half of NP. All phosphothreonine residues and consensus recognition sequences for protein kinase CKII are located in regions I*-V*. Regions VI* and VII* contain only phosphoserine with three of four serine residues in consensus recognition motifs for proline-directed protein kinases. Mutagenesis of proline-adjacent serine residues to alanine or aspartic acid did not influence the function of NP in a reconstituted transcription/replication system; thus it is concluded that serine phosphorylation in the most C-terminal part of NP is not a regulatory factor in viral RNA synthesis.
Insights
Marburg virus nucleoprotein (NP) phosphorylation occurs primarily on serine and threonine residues in the C-terminal half. Specific serine modifications in the C-terminal regions do not impact viral RNA synthesis.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- The Marburg virus nucleoprotein (NP) is crucial for viral RNA synthesis.
- Understanding NP post-translational modifications, like phosphorylation, is key to deciphering viral replication mechanisms.
Purpose of the Study:
- To identify and characterize the phosphorylation sites on Marburg virus nucleoprotein (NP).
- To investigate the functional relevance of NP phosphorylation in viral RNA synthesis.
Main Methods:
- Analysis of phosphoamino acid content and phosphorylation sites in NP from virions and expression systems.
- Expression of truncated NPs in HeLa cells using the vaccinia virus T7 system.
- Site-directed mutagenesis of identified phosphorylation sites.
- Assessment of NP function in a reconstituted transcription/replication system.
Main Results:
- Marburg virus NP is phosphorylated at serine and threonine residues (85:15 ratio); phosphotyrosine is absent.
- Phosphorylation is confined to the C-terminal half of NP, with seven identified phosphorylated regions (I*-VII*).
- Phosphothreonine residues and CKII kinase motifs are in regions I*-V*; phosphoserine and proline-directed kinase motifs are in regions VI*-VII*.
- Mutagenesis of specific serine residues in the C-terminal regions did not affect NP function in transcription/replication.
Conclusions:
- Marburg virus NP phosphorylation is predominantly localized to the C-terminal half.
- Serine phosphorylation in the C-terminal regions of NP is not a regulatory factor for viral RNA synthesis.