| dc.description.abstract | Mapping of Interacting Domains of Rinderpest Virus Nucleocapsid Protein and Phosphoprotein Involved in Transcription and Replication
Introduction
Rinderpest is a highly contagious disease of cattle and wild bovids, with morbidity and mortality rates approaching 95%. The causative agent, Rinderpest virus (RPV), is a negative-stranded RNA virus belonging to the genus Morbillivirus in the family Paramyxoviridae.
The RPV genome is 15,882 bases long and contains a 52-base leader RNA at the 3 end, followed by coding sequences for structural proteins in the order: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), surface glycoproteins (F and H), and the large polymerase protein (L). The P gene also encodes two non-structural proteins, C and V. The C protein is expressed from an overlapping reading frame, while the V protein is generated by RNA editing at a specific site.
The viral genome is tightly encapsidated by the N protein, forming the N-RNA complex, which serves as the template for transcription and replication. The transcription-replication complex consists of N (525 amino acids), P (508 amino acids), and L (2183 amino acids). These proteins interact through specific domains to mediate viral transcription and replication.
Key Findings
1. Self-Association of RPV P Protein
Yeast two-hybrid analysis of 16 P protein deletions revealed that a 32-amino acid region (316-347 aa) at the C-terminus mediates self-association.
This region has a high probability of forming coiled coils, suggesting that P protein self-association is coiled coil-mediated.
The coiled coil region alone was sufficient to activate the -galactosidase reporter gene in yeast assays.
2. Domains of P Protein Involved in N-P Complex Formation
Co-expression of N and P proteins prevents N self-assembly, forming soluble N°-P complexes.
Mapping revealed that both the N-terminal 59 amino acids and the coiled coil region of P are required for N°-P complex formation.
This suggests that oligomeric P proteins contribute multiple N-binding pockets.
The C protein showed no interaction with N or P, implying its inhibitory role may involve L protein.
3. Domains of N Protein Contributing to N-P Complex Formation
Two independent domains in the C-terminal half of N protein (335-392 aa and 393-525 aa) were identified.
Together, these domains synergistically enhanced N-P complex formation.
The C-terminal 335-525 aa fragment was sufficient to bind the minimal P protein region required for N-P interaction.
4. N Protein Self-Assembly
Expression of N protein in E. coli resulted in multimerization and formation of nucleocapsid-like structures.
Deletion analysis showed that the conserved N-terminal 391 amino acids are responsible for nucleocapsid assembly.
5. Interaction of Assembled N Protein with P Protein
Glycerol pelleting assays revealed that the C-terminal 46 amino acids of assembled N protein mediate binding to P protein.
This interaction is critical for recognition of the N-RNA template by L protein during transcription and replication.
6. P Protein Interaction with N-RNA Template
N proteins expressed alone encapsidated E. coli RNA to form nucleocapsid-like particles.
These particles specifically bound recombinant P protein, independent of phosphorylation.
Mapping showed that both the coiled coil region and the extreme C-terminal 17 amino acids of P are required for nucleocapsid binding.
Results suggest oligomeric P proteins bind nucleocapsids through multiple C-terminal regions.
Conclusion
This study provides a comprehensive mapping of interacting domains of RPV N and P proteins involved in transcription and replication. The findings highlight:
Coiled coil-mediated self-association of P protein.
Dual-domain contributions of N protein to N-P complex formation.
Critical roles of N-terminal and C-terminal regions in nucleocapsid assembly and P binding.
Essential involvement of P protein’s coiled coil and C-terminal regions in nucleocapsid interactions.
These insights enable a clearer visualization of N-P protein interactions and provide a foundation for further functional analysis of viral transcription and replication mechanisms. | |
