Domains of Rinderpest virus phosphoprotein involved in interaction with itself and the nucleocapsid protein

The yeast two-hybrid system was used to identify domains involved in specif ic in vivo interactions between the Rinderpest virus (RPV) phosphoprotein ( P) and nucleocapsid protein (N). N and P genes were cloned in both the yeas t GAL4 DNA-binding and GAL4 activation domain vectors, which enabled analys is of self and interprotein interactions. Mapping of the domain of P protei n involved in its association with itself revealed that the COOH-terminal 3 2 amino acids (316-347) that forms a part of the highly conserved coiled co il region is important for interaction. In addition, just the coiled coil r egion of RPV P protein fused to the DNA-binding domain and activation domai n bf GAL4 was found to be sufficient to bring about activation of the beta- galactosidase reporter. Similarly, mapping of the domains of P protein invo lved 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 interactio n are simultaneously required for association with N protein. Interestingly , a P protein mutant with just The NH2-terminal 59 amino acids and the coil ed coil domain with all other P protein regions deleted retained its abilit y to interact with N protein. Furthermore, we were able to show IV and P pr otein interaction in vitro using recombinant N and P proteins expressed in Escherichia coli, demonstrating the existence of direct physical interactio n between the two proteins. (C) 1999 Academic Press.