Domains of Rinderpest Virus Phosphoprotein Involved in Interaction with Itself and the Nucleocapsid Protein

• Published in: Virology (New York, N.Y.) Vol. 258; no. 2; pp. 415 - 424
• Main Authors: Shaji, Daniel, Shaila, M.S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.06.1999
• Subjects: animal diseases; animal health; Animals; Binding Sites; interaction; nucleocapsid protein; Nucleocapsid Proteins - genetics; Nucleocapsid Proteins - metabolism; phosphoprotein; Phosphoproteins - genetics; Phosphoproteins - metabolism; Rabbits; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Rinderpest virus; Rinderpest virus - genetics; Rinderpest virus - metabolism; viral diseases of animals and humans; Viral Proteins - genetics; Viral Proteins - metabolism; yeast two-hybrid system; yeast two-hybrid system; interaction; nucleocapsid protein; Rinderpest virus; phosphoprotein
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1006/viro.1999.9740

The yeast two-hybrid system was used to identify domains involved in specific in vivo interactions between the Rinderpest virus (RPV) phosphoprotein (P) and nucleocapsid protein (N). N and P genes were cloned in both the yeast GAL4 DNA-binding and GAL4 activation domain vectors, which enabled analysis of self and interprotein interactions. Mapping of the domain of P protein involved in its association with itself revealed that the COOH-terminal 32 amino acids (316–347) that forms a part of the highly conserved coiled coil region is important for interaction. In addition, just the coiled coil region of RPV P protein fused to the DNA-binding domain and activation domain of GAL4 was found to be sufficient to bring about activation of the β-galactosidase reporter. Similarly, mapping of the domains of P protein involved in its interaction with N protein revealed that NH2-terminal 59 amino acids and COOH-terminal 32 amino acids (316–347) involved in P–P interaction are simultaneously required for association with N protein. Interestingly, a P protein mutant with just the NH2-terminal 59 amino acids and the coiled coil domain with all other P protein regions deleted retained its ability to interact with N protein. Furthermore, we were able to show N and P protein interaction in vitro using recombinant N and P proteins expressed in Escherichia coli, demonstrating the existence of direct physical interaction between the two proteins.