ABSTRACT The ambisense RNA genome of Tomato spotted wilt virus (TSWV) isby interaction with numerous copies of the virus encoded nucleocapsid (N) protein to form a subvirion structure called a ribonucleo-protein (RNP). RNPs are central to the viral replication cycle because they, and not free viral RNA, serve as templates for viral gene expression and genome replication. N protein monomers bind to viral RNA molecules in a cooperative manner. We have examined regions of the N protein that are involved in the N-N interactions that likely contribute to the cooperative binding of N to viral RNA. We created random and alanine scanning mutants of N and then screened the mutants for defects in N-N interaction using reverse and forward yeast two-hybrid assays. Our experiments identified residues in three distinct regions of the primary structure of the protein, residues 42 to 56, 132 to 152, and in the C-terminal 26 amino acids, that contribute to N-N dimerization or multimerization.interactions between N monomers mediated by the residues we identified are of a nonelectrostatic nature.