The Replication Function of Rabies Virus P Protein Is Regulated by a Novel Phosphorylation Site in the N-Terminal N Protein-Binding Region
Abstract
1. Introduction
2. Methods
2.1. Plasmids
2.2. Cell Culture, Transfection and Treatments
2.3. Luciferase Reporter Gene Assays
2.4. Cell Imaging
2.5. Mass Spectrometry
2.6. Western Analysis of Cell Lysates
2.7. Protein Expression and Purification
2.8. Protein Crystallisation, Data Collection, and Processing
2.9. Statistical Analysis
3. Results and Discussion
3.1. P Protein Is Phosphorylated in Multiple Regions Including at Novel Sites
3.2. Modification of Selected Phosphorylation Sites Does Not Substantially Alter P1 Nuclear Localization
3.3. Modification of Selected Phosphorylation Sites Does Not Significantly Impact on IFN Antagonist Functions of P1
3.4. S48E Impacts on P Protein Function in Transcription/Replication
3.5. Modification at Ser48 Impacts on the Formation of Negri Body-like Structures
3.6. Expression and Crystallisation of Recombinant N0-P1−52 Complexes
3.7. Crystal Structures of Ni-CE N0-P and Ni N0-P-S48E
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Data Collection and Processing | Ni-CE N0P | Ni N0P-S48E |
---|---|---|
Wavelength (Å) | 0.9537 | 0.9537 |
Resolution range (Å) | 29.90–2.00 (2.05–2.00) | 29.88–2.10 (2.16–2.10) |
Space group | P 21 21 21 | P 21 21 21 |
Unit cell (Å, °) | 42.17 74.50 154.72, 90 90 90 | 41.89 75.39 154.99, 90 90 90 |
Total reflections | 388,562 (22,768) | 138,385 (10,791) |
Unique reflections | 33,217 (2309) | 29,538 (2335) |
Multiplicity | 11.7 (9.9) | 4.7 (4.6) |
Completeness (%) | 98.1 (95.1) | 99.9 (99.8) |
Mean I/sigma(I) | 12.4 (1.8) | 5.9 (2.0) |
Wilson B-factor Å2 | 23.177 | 21.811 |
R-merge % | 0.141 (1.380) | 0.138 (0.854) |
R-pim % | 0.042 (0.428) | 0.071 (0.456) |
CC1/2 | 0.997 (0.656) | 0.989 (0.849) |
Refinement | ||
Resolution range (Å) | 20.0–2.0 | 20.0–2.15 |
Number of reflections | 33,008 | 27,965 |
Number of R-free reflections | 1718 | 1474 |
R-work % | 18.22 | 18.28 |
R-free | 21.70 | 21.48 |
RMS (bonds) | 0.003 | 0.003 |
RMS (angles) | 0.60 | 0.58 |
Ramachandran plot | ||
favoured (%) | 98.26 | 98.10 |
allowed (%) | 1.74 | 1.90 |
outliers (%) | 0 | 0 |
Validation | ||
Clash score | 0.62 | 2.22 |
MolProbity score (percentile) | 0.71 (100) | 1.00 (100) |
PDB accession code | 8U0A | 8U0B |
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Tudhope, E.; Donnelly, C.M.; Sethi, A.; David, C.; Williamson, N.; Stewart, M.; Forwood, J.K.; Gooley, P.R.; Moseley, G.W. The Replication Function of Rabies Virus P Protein Is Regulated by a Novel Phosphorylation Site in the N-Terminal N Protein-Binding Region. Viruses 2025, 17, 1075. https://doi.org/10.3390/v17081075
Tudhope E, Donnelly CM, Sethi A, David C, Williamson N, Stewart M, Forwood JK, Gooley PR, Moseley GW. The Replication Function of Rabies Virus P Protein Is Regulated by a Novel Phosphorylation Site in the N-Terminal N Protein-Binding Region. Viruses. 2025; 17(8):1075. https://doi.org/10.3390/v17081075
Chicago/Turabian StyleTudhope, Ericka, Camilla M. Donnelly, Ashish Sethi, Cassandra David, Nicholas Williamson, Murray Stewart, Jade K. Forwood, Paul R. Gooley, and Gregory W. Moseley. 2025. "The Replication Function of Rabies Virus P Protein Is Regulated by a Novel Phosphorylation Site in the N-Terminal N Protein-Binding Region" Viruses 17, no. 8: 1075. https://doi.org/10.3390/v17081075
APA StyleTudhope, E., Donnelly, C. M., Sethi, A., David, C., Williamson, N., Stewart, M., Forwood, J. K., Gooley, P. R., & Moseley, G. W. (2025). The Replication Function of Rabies Virus P Protein Is Regulated by a Novel Phosphorylation Site in the N-Terminal N Protein-Binding Region. Viruses, 17(8), 1075. https://doi.org/10.3390/v17081075