Mapping of interacting domains of rinderpest virus nucleaocapsid protein and phospoprotein involved in transcription and Replication

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.