A HIGHLY CONSERVED REGION OF THE SENDAI VIRUS NUCLEOCAPSID PROTEIN CONTRIBUTES TO THE NP-NP BINDING DOMAIN

The nucleocapsid protein (NP) of Sendai virus is an essential componen t of both the nucleocapsid template and the NP-NP and NP0-P protein co mplexes required for viral RNA replication. When expressed alone in ma mmalian cells NP self-assembles into nucleocapsidlike particles which appear to contain cellular RNA. To identify putative NP-NP binding dom ains, fusions between the monomeric maltose-binding protein (MBP) and portions of NP were constructed. The fusion proteins which contain the central conserved region (CCR) (amino acids 258-357, MBP-NP1) and the N-terminal 255 amino acids (MBP-MP2) of NP both oligomerized, suggest ing that these regions contain sequences important for NP-NP self-asse mbly. in addition, the MBP-NP1 fusion protein can function as an inhib itor of viral RNA replication. Complementary studies involving site-di rected mutagenesis of the full-length MP protein have identified speci fic residues in the CCR which are essential for viral RNA replication in vitro. Two such replication-negative mutants, F324V and F324I, were defective in self-assembly, suggesting that the Phe residue at amino acid 324 is essential for the NP-NP interaction. A third mutant, NP260 -1 (Y260D), self-assembled to form aberrant oligomers which exhibit an unusual helical structure and appear to lack any associated RNA. The mutants NP299-5 (L299I and I300V) and NP313-2 (I313F), in contrast, ap pear to form all the required protein complexes, but were inactive in viral RNA replication, suggesting that interactions specifically with Sendai RNA were disrupted. These data have thus identified specific re sidues in the CCR of the native NP protein which appear to be importan t for NP-NP or NP-RNA interactions and for genome replication. (C) 199 7 Academic Press.